Bioconjugates made from recombinant N-glycosylated proteins from procaryotic cells

ABSTRACT

The present invention is directed to a bioconjugate vaccine, such as an O1-bioconjugate vaccine, comprising: a protein carrier comprising a protein carrier containing at least one consensus sequence, D/E-X-N-Z-S/T, wherein X and Z may be any natural amino acid except proline; at least one antigenic polysaccharide from at least one pathogenic bacterium, linked to the protein carrier; and, optionally, an adjuvant. In another aspect, the present invention is directed to a method of producing an O1-bioconjugate in a bioreactor comprising a number steps.

This application is a continuation of U.S. patent application Ser. No.14/735,773, filed Dec. 14, 2010, which is a U.S. national stage entry ofInternational Patent Application No. PCT/IB2009/000287, filed Feb. 19,2009, which claims benefit of U.S. Provisional Patent Application Nos.61/064,163, filed Feb. 20, 2008; 61/071,545, filed May 5, 2008;61/129,480, filed Jun. 30, 2008; 61/129,852, filed Jul. 24, 2008; and61/136,687, filed Sep. 25, 2008, each of which are incorporated hereinby reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to bioconjugates, specificallybioconjugate vaccines, made from recombinant glycoproteins, namelyN-glycosylated proteins. The invention comprises one or more introducedN-glycosylated proteins with optimized amino acid consensus sequence(s),nucleic acids encoding these proteins as well as corresponding vectorsand host cells. In addition, the present invention is directed to theuse of said proteins, nucleic acids, vectors and host cells forpreparing bioconjugate vaccines. Furthermore, the present inventionprovides methods for producing bioconjugate vaccines.

BACKGROUND OF THE INVENTION

Glycoproteins are proteins that have one or more covalently attachedsugar polymers. N-linked protein glycosylation is an essential andconserved process occurring in the endoplasmic reticulum of eukaroticorganisms. It is important for protein folding, oligomerization,stability, quality control, sorting and transport of secretory andmembrane proteins (Helenius, A., and Aebi, M. (2004). Roles of N-linkedglycans in the endoplasmic reticulum. Annu. Rev. Biochem. 73,1019-1049).

Protein glycosylation has a profound influence on the antigenicity, thestability and the half-life of a protein. In addition, glycosylation canassist the purification of proteins by chromatography, e.g. affinitychromatography with lectin ligands bound to a solid phase interactingwith glycosylated moieties of the protein. It is therefore establishedpractice to produce many glycosylated proteins recombinantly ineukaryotic cells to provide biologically and pharmaceutically usefulglycosylation patterns.

It has been demonstrated that a bacterium, the food-borne pathogenCampylobacter jejuni, can also N-glycosylate its proteins (Szymanski, etal. (1999). Evidence for a system of general protein glycosylation inCampylobacter jejuni. Mol. Microbiol. 32, 1022-1030). The machineryrequired for glycosylation is encoded by 12 genes that are clustered inthe so-called pgl locus. Disruption of N-gylcosylation affects invasionand pathogenesis of C. jejuni but is not lethal as in most eukaryoticorganisms (Burda P. and M. Aebi, (1999). The dolichol pathway ofN-linked glycosylation. Biochim Biophys Acta 1426(2):239-57). It ispossible to reconstitute the N-glycosylation of C. jejuni proteins byrecombinantly expressing the pgl locus and acceptor glycoprotein in E.coli at the same time (Wacker et al. (2002). N-linked glycosylation inCampylobacter jejuni and its functional transfer into E. coli. Science298, 1790-1793).

Diarrheal illness is a major health problem associated withinternational travel in terms of frequency and economic impact.Traveller's diarrhea refers to an enteric illness acquired when a persontravels from a developed to a developing country. Today, over 50 millionpeople travel each year from developed countries to developing countriesand up to 50% of these travelers report having diarrhea during the first2 weeks of their week of their stay. There has been no significantdecline in the incidence of traveller's diarrhea since the 1970s,despite efforts made by the tourism industry to improve localinfrastructure.

Traveller's diarrhea is acquired through the ingestion of faecallycontaminated food and less commonly water. Bacteria are the main causeof traveller diarrhea's, being responsible for up to 80% of theinfections. Enterotoxigenic E. coli(ETEC) is the most frequentlyisolated bacterium in all parts of the world associated with traveler'sdiarrhea, followed by Shigella spp and C. jejuni.

Shigellosis remains a serious and common disease. In addition to causingwatery diarrhea, Shigellae are a major cause of dysentery (fever,cramps, and blood and/or mucus in the stool). Man is the only naturalhost for this bacterium. The estimated number of Shigella infections isover 200 million annually. About 5 million of these cases needhospitalization and a million people die. Three serogroups are mostlyresponsible for the disease described as bacillary dysentery: S.dysenteriae, S. flexneri and S. sonnei.

S. dysenteriae and S. flexneri are responsible for most infections inthe tropics, with case fatalities up to 20%. Shigellosis occurs bothendemically and as epidemics. In many tropical countries, endemicinfection is largely due to S. flexneri whereas major epidemics of S.dysenteriae have occurred in Central America, Central Africa andSoutheast Asia. These epidemics are major public-health risks.Infections, primarily due to S. sonnei and less frequently flexnericontinue to occur in industrialized countries.

Conjugate vaccines have shown promising results against Shigellainfections. O-specific polysaccharides of S. dysenteriae type 1 havebeen used to synthesize a conjugate vaccine that has elicited an immuneresponse in mice. Such vaccines have been synthesized chemically andconjugated to human serum albumin or has been developed where theO-polysaccharide has been purified from Shigella. The O-specificpolysaccharides of S. sonnei and S. flexneri also have been conjugatedchemically to P. aeruginosa exotoxin and have elicited a significantimmune response in mice. Additionally, they have been shown to beimmunogenic and safe in humans. However, chemical conjugation is anexpensive and time-consuming process that does not always yield reliableand reproducible vaccines. This leads to good manufacturing practices(GMP) problems when seeking to develop such bioconjugate vaccines on acommercial scale.

SUMMARY OF THE INVENTION

In one aspect, the present invention is directed to a bioconjugatevaccine comprising: a protein carrier comprising an inserted consensussequence, D/E-X-N-Z-S/T, wherein X and Z may be any natural amino acidexcept proline; at least one antigenic polysaccharide from at least onebacterium, linked to the protein carrier, wherein the at least oneantigenic polysaccharide is at least one bacterial O-antigen from one ormore strains of Shigella, E. coli or Pseudomonas aeruginosa; and,optionally, an adjuvant.

In another aspect, the present invention is directed to a Shigellabioconjugate vaccine comprising: a protein carrier comprising Exotoxinof Pseudomonas aeruginosa (EPA) that has been modified to contain atleast one consensus sequence D/E-X-N-Z-S/T, wherein X and Z may be anynatural amino acid except proline; at least one polysaccharide chainlinked to the protein carrier and having the following structure:

and, optionally, an adjuvant.

In yet another aspect, the present invention is directed to a Shigelladysenteriae O1 bioconjugate vaccine comprising: a protein carrier havingthe sequence provided in SEQ. ID NO.: 7; at least one polysaccharidechain linked to the protein carrier and having the following structure:

and an adjuvant.

In yet additional aspects, the present invention is directed to: aplasmid comprising SEQ. ID NO. 5; a genetic sequence comprising SEQ. IDNO. 5; an amino acid sequence comprising SEQ. ID NO. 6; an amino acidsequence comprising SEQ. ID NO. 7; or vector pGVXN64.

In another aspect, the present invention is directed to an expressionsystem for producing a bioconjugate vaccine against at least onebacterium comprising: a nucleotide sequence encoding an oligosaccharyltransferase (OST/OTase); a nucleotide sequence encoding a proteincarrier; and at least one antigenic polysaccharide synthesis genecluster from the at least one bacterium, wherein the antigenicpolysaccharide is a bacterial O-antigen.

In still another aspect, the present invention is directed to anexpression system for producing a bioconjugate vaccine against Shigelladysenteriae O1 comprising: a nucleotide sequence encoding PgIB havingSEQ. ID NO. 2; a nucleotide sequence encoding a modified EPA having SEQ.ID NO. 6; and a polysaccharide synthesis gene cluster comprising SEQ. IDNO. 5.

In yet another aspect, the present invention contemplates a method ofproducing an O1-bioconjugate in a bioreactor comprising the steps:expressing in bacteria: modified EPA containing at least one consensussequence, D/E-X-N-Z-S/T, wherein X and Z may be any natural amino acidexcept proline, or AcrA; PgIB; and one or more O1-polysaccharides;growing the bacteria for a period of time to produce an amount of theO1-bioconjugate comprising the AcrA or the modified EPA linked to theone more O1-polysaccharides; extracting periplasmic proteins; andseparating the O1-bioconjugate from the extracted periplasmic proteins.

In an additional aspect, the present invention contemplates a method ofproducing an S. dysenteriae bioconjugate vaccine, said methodcomprising: assembling a polysaccharide of S. dysenteriae in arecombinant organism through the use of glycosyltransferases; linkingsaid polysaccharide to an asparagine residue of one or more targetproteins in said recombinant organism, wherein said one or more targetproteins contain one or more T-cell epitopes.

In a further aspect, the present invention contemplates a method ofproducing an S. dysenteriae bioconjugate vaccine, said methodcomprising: introducing genetic information encoding for a metabolicapparatus that carries out N-glycosylation of a target protein into aprokaryotic organism to produce a modified prokaryotic organism, whereinthe genetic information required for the expression of one or morerecombinant target proteins is introduced into said prokaryoticorganism, and wherein the metabolic apparatus comprises specificglycosyltransferases for the assembly of a polysaccharide of S.dysenteriae on a lipid carrier and an oligosaccharyltransferase, theoligosaccharyltransferase covalently linking the polysaccharide to anasparagine residue of the target protein, and the target proteincontaining at least one T-cell epitope; producing a culture of themodified prokaryotic organism; and obtaining glycosylated proteins fromthe culture medium.

DESCRIPTION OF THE FIGURES

FIG. 1 illustrates the N-glycosylation of Lip proteins derived fromconstructs A to C (see example 1). E. coli Top 10 cells carrying afunctional pgl operon from C. jejuni (Wacker et al., 2002, supra) and aplasmid coding for constructs A (lane 2), B (lane 1), and C (lane 3) ora mutant of construct C with the mutation D121A (lane 4). Proteins wereexpressed and purified from periplasmic extracts. Shown is the SDS-PAGEand Coomassie brilliant blue staining of the purified protein fractions.

FIG. 2 shows the N-glycosylation analysis of the different proteins thatwere analyzed for the sequence specific N-glycosylation by the C. jejunipgl operon (Wacker et al., 2002, supra) in CLM24 cells (Feldman et al.,(2005). Engineering N-linked protein glycosylation with diverse Oantigen lipopolysaccharide structures in Escherichia coli. Proc. Natl.Acad. Sci. USA 102, 3016-3021) or Top10 cells (panel E lanes 1-6) orSCM7 cells (Alaimo, C, Catrein, I., Morf, L., Marolda, C. L.,Callewaert, N., Valvano, M. A., Feldman, M. F., Aebi, M. (2006). Twodistinct but interchangeable mechanisms for flipping of lipid-linkedoligosaccharides. EMBO Journal 25, 967-976) (panel E, lanes 7, 8)expressing said proteins from a plasmid. Shown are SDS-PAGE separatedperiplasmic extracts that were transferred to a nitrocellulose membraneand visualized with specific antisera. In panels A-D, the top panelsshow immunoblots probed with anti AcrA antiserum (Wacker et al. 2002,supra; Nita-Lazar, M., Wacker, M., Schegg, B., Amber, S., and Aebi, M.(2005). The N-X-S/T consensus sequence is required but not sufficientfor bacterial N-linked protein glycosylation. Glycobiology 15, 361-367),whereas the bottom panels show immunoblots probed with R12 antiserum(Wacker et al., 2002, supra). + and − indicate the presence of thefunctional or mutant pgl operon in the cells. Panel A contains samplesof the soluble wildtype AcrA with the pelB signal sequence and the hexahistag (lanes 1, 2), AcrA-N273Q (lane 3, 4), and AcrA-D121A (lane 5).Panel B: AcrA (lanes 1, 2), AcrA-T145D (lane 3), AcrA-N123Q-N273Q-T145D(lanes 4, 5). Panel C: AcrA-F115D-T145D (lanes 1, 2),AcrA-N123Q-N273Q-N272D (lanes 3, 4). Panel D: AcrA-N273Q (lanes 1, 2),AcrA-N273Q-F122P (lanes 3, 4). Panel E: CtxB (lanes 1, 2), CtxB-W88D(lanes 3, 4), CtxB-Q56/DSNIT (lanes 5, 6), and CtxB-W88D-Q56/DSNIT.

FIG. 3 shows the engineering of multiple glycosylation sites in OmpH1.The ΔwaaL strain SCM6 was co-transformed with plasmid pACYCpgl (encodingentire pgl locus) and plasmids expressing wild type OmpH1 (lane 1),OmpH1^(N139S)-myc (lane 2), OmpH1^(KGN→NIT HFGDD→DSNIT)-myc (lane 3),QmpH1^(RGD→NIT, HFGDD→DSNT)-myc (|lane 4), OmpH1^(KGN→NIT, RGD→NIT)-myc(lane 5)^(KGN-NIT, HFGDD→DSNIT)-myc (lane 6) orOmpH1^(RGD→NIT, V83T)-myc (lane 7). The cells were grown aerobically,induced with 0.5% arabinose for 3 hours prior to analysis. Whole celllysates were TCA precipitated after equalizing the optical density ofthe cultures as described in the materials and methods section. Theproteins were separated by 15% SDS-PAGE and transferred onto a PVDFmembrane. First panel, immunoblot of whole cell lysates probed withanti-myc tag antobodies. Bottom panel, immunoblot of whole cell lysatesprobed with glycan-specific antiserum. The positions of unglycosylated-and glycosylated OmpH1 are indicated on the right.

FIG. 4. shows fluorescence microscopy of cells expressing various OmpH1variants. Fluorescence microscopy was performed by the using anAxioplan2 microscope (Carl Zeiss). Images were combined by using AdobePhotoshop, version CS2. SCM6 cells expressing OmpH1 (panel A),OmpH1^(N139S) (panel B), OmpH1^(C20)S (panel C),OmpH1^(KGN→NIT,HFGDD→DSNIT) (panel D), OmpH1^(RGD→NIT,) ^(HFGDD→DSNIT)(panel E), OmpH1^(KGN→NIT RGD→NIT) (panel F), OmpH1^(V83T,KGN→NIT)(panel G), and OmpH1^(KGN→NIT,) ^(RGD→NIT,) ^(HFGDD→DSNIT) (panel H).The first column is a merge of the pictures in columns 2, 3, and 4represented in greytones on black background. Column 2: bluefluorescence in greytones from DAPI stain, column 3: green fluorescencefrom glycan specific fluorescence, column 4: red fluorescence fromanti-myc staining.

FIG. 5A shows a schematic of the capsular polysaccharides andlipopolysacharides in Gram-positive and Gram-negative bacteria.

FIG. 5B shows genomic DNA, with integrated PgIB and EPA, and plasmidDNA, which is interchangeable (i.e., exchangeable), encoding apolysaccharide synthesis gene cluster.

FIG. 6A shows the production process of conjugate vaccines usingtechnology of the invention.

FIG. 6B shows the construction of the Shigella dysenteriae O1 antigenexpression plasmid pGVXN64.

FIGS. 7A and 7B show schematics of the protein glycosylation pathwayutilized in the present invention.

FIGS. 8A and 8B are schematics depicting expression platforms forbioconjugate production of the present invention.

FIG. 9 shows production of Shigella bioconjugates.

FIG. 10A shows polysaccharide biosynthesis of the O-antigen of S.dysenteriae Serotype O1 on undecaprenolpyrophosphate (UPP).

FIG. 10B shows a schematic of a carrier protein, such as EPA, onto whichN-glycosylation sites can be designed.

FIG. 11 shows bicoonjugates that elicit an immune response againstShigella O1 polysaccharide in mice.

FIG. 12 shows results from the production of a Shigella O1-EPAbioconjugate (e.g., O1-EPA) in a bioreactor.

FIG. 13 shows purification of O1-EPA.

FIG. 14A shows a Western blot analysis of a series of specimens ofShigella O1-AcrA bioconjugates produced in an LB shake flask taken undervarious conditions.

FIG. 14B provides different serotypes of Shigella and the polysaccharidestructure that defines their antigenicity (i.e., Shigella O-antigens).

FIG. 15 shows the expansion of the anomeric region of 1H NMR spectrum ofan example of a S. dysenteriae Serotype O1 bioconjugate of theinvention.

FIG. 16A shows protein samples of a Shigella O1 Bioconjugate (e.g.,EPA-O1) normalized to biomass concentration (0.1 OD_(600nm) ofcells/lane).

FIG. 16B shows the periplasmic extract from a Shigella O1 Bioconjugateproduction process which was loaded on a 7.5% SDS-PAGE, and stained withCoomasie to identify EPA and EPA-O1.

FIG. 17A shows protein fractions from 1. Source Q analyzed by SDS-PAGEand stained by Coomassie to identify the Shigella O1 bioconjugate.

FIG. 17B shows protein fractions from 2. Source Q column analyzed onSDS-PAGE and stained by Coomassie to identify the Shigella O1bioconjugate.

FIG. 18A shows protein fractions from Superdex 200 column analyzed bySDS-PAGE and stained by Coomassie stained to identify the Shigella O1bioconjugate.

FIG. 18B shows Shigella bioconjugates from different purification stepsanalyzed by SDS-PAGE and stained by Coomassie.

DETAILED DESCRIPTION OF THE INVENTION Introduction to Invention

The present invention provides a versatile in vivo glycosylationplatform.

European Patent Application No. 03 702 276.1 (European Patent 1 481 057)teaches a procaryotic organism into which is introduced a nucleic acidencoding for (i) specific glycosyltransferases for the assembly of anoligosaccharide on a lipid carrier, (ii) a recombinant target proteincomprising a consensus sequence “N-X-S/T”, wherein X can be any aminoacid except proline, and (iii) an oligosaccharyl transferase of C.jejuni (OTase) that covalently links said oligosaccharide to theconsensus sequence of the target protein. Said procaryotic organismproduces N-glycans with a specific structure which is defined by thetype of the specific glycosyltransferases.

The known N-glycosylation consensus sequence in a protein allows for theN-glycosylation of recombinant target proteins in procaryotic organismscomprising the oligosaccharyl transferase (OTase) of C. jejuni.

The object of the present invention is to provide proteins as well asmeans and methods for producing such proteins having an optimizedefficiency for N-glycosylation that can be produced in procaryoticorganisms in vivo. Another object of the present invention aims at themore efficient introduction of N-glycans into recombinant proteins formodifying antigenicity, stability, biological, prophylactic and/ortherapeutic activity of said proteins. A further object is the provisionof a host cell that efficiently displays recombinant N-glycosylatedproteins of the present invention on its surface.

In a first aspect, the present invention provides a recombinantN-glycosylated protein, comprising one or more of the followingN-glycosylated optimized amino acid sequence(s):D/E-X-N-Z-S/T, (optimized consensus sequence)wherein X and Z may be any natural amino acid except Pro, and wherein atleast one of said N-glycosylated partial amino acid sequence(s) isintroduced.

It was surprisingly found that the introduction of specific partialamino acid sequence(s) (optimized consensus sequence(s)) into proteinsleads to proteins that are efficiently N-glycosylated by theoligosaccharyl transferase (OST, OTase) from Campylobacter spp.,preferably C. jejuni, in these introduced positions.

The term “partial amino acid sequence(s)” as it is used in the contextof the present invention will also be referred to as “optimizedconsensus sequence(s)” or “consensus sequence(s)”. The optimizedconsensus sequence is N-glycosylated by the oligosaccharyl transferase(OST, OTase) from Campylobacter spp., preferably C. jejuni, much moreefficiently than the regular consensus sequence “N-X-SIT” known in theprior art.

In general, the term “recombinant N-glycosylated protein” refers to anyheterologous poly- or oligopeptide produced in a host cell that does notnaturally comprise the nucleic acid encoding said protein. In thecontext of the present invention, this term refers to a protein producedrecombinantly in any host cell, e.g. an eukaryotic or prokaryotic hostcell, preferably a procaryotic host cell, e.g. Escherichia ssp.,Campylobacter ssp., Salmonella ssp., Shigella ssp., Helicobacter ssp.,Pseudomonas ssp., Bacillus ssp., more preferably Escherichia coli,Campylobacter jejuni, Salmonella typhimurium etc., wherein the nucleicacid encoding said protein has been introduced into said host cell andwherein the encoded protein is N-glycosylated by the OTase fromCampylobacter spp., preferably C. jejuni, said transferase enzymenaturally occurring in or being introduced recombinantly into said hostcell.

In accordance with the internationally accepted one letter code foramino acids the abbreviations D, E, N, S and T denote aspartic acid,glutamic acid, asparagine, serine, and threonine, respectively. Proteinsaccording to the invention differ from natural or prior art proteins inthat one or more of the optimized consensus sequence(s) D/E-X-N-Z-S/Tis/are introduced and N-glycosylated. Hence, the proteins of the presentinvention differ from the naturally occurring C. jejuni proteins whichalso contain the optimized consensus sequence but do not comprise anyadditional (introduced) optimized consensus sequences.

The introduction of the optimized consensus sequence can be accomplishedby the addition, deletion and/or substitution of one or more aminoacids. The addition, deletion and/or substitution of one or more aminoacids for the purpose of introducing the optimized consensus sequencecan be accomplished by chemical synthetic strategies well known to thoseskilled in the art such as solid phase-assisted chemical peptidesynthesis. Alternatively, and preferred for larger polypeptides, theproteins of the present invention can be prepared by standardrecombinant techniques.

The proteins of the present invention have the advantage that they maybe produced with high efficiency and in any procaryotic host comprisinga functional pgl operon from Campylobacter spp., preferably C. jejuni.Preferred alternative OTases from Campylobacter spp. for practicing theaspects and embodiments of the present invention are Campylobacter coliand Campylobacter lari (see Szymanski, C. M. and Wren, B. W. (2005).Protein glycosylation in bacterial mucosal pathogens. Nat. Rev.Microbiol. 3:225-237). The functional pgl operon may be presentnaturally when said procaryotic host is Campylobacter spp., preferablyC. jejuni. However, as demonstrated before in the art and mentionedabove, the pgl operon can be transferred into cells and remainfunctional in said new cellular environment.

The term “functional pgl operon from Campylobacter spp., preferably C.jejuni” is meant to refer to the cluster of nucleic acids encoding thefunctional oligosaccharyl transferase (OTase) of Campylobacter spp.,preferably C. jejuni, and one or more specific glycosyltransferasescapable of assembling an oligosaccharide on a lipid carrier, and whereinsaid oligosaccharide can be transferred from the lipid carrier to thetarget protein having one or more optimized amino acid sequence(s):D/E-X N-Z-S/T by the OTase. It to be understood that the term“functional pgl operon from Campylobacter spp., preferably C. jejuni’ inthe context of this invention does not necessarily refer to an operon asa singular transcriptional unit. The term merely requires the presenceof the functional components for N-glycosylation of the recombinantprotein in one host cell. These components may be transcribed as one ormore separate mRNAs and may be regulated together or separately. Forexample, the term also encompasses functional components positioned ingenomic DNA and plasmid(s) in one host cell. For the purpose ofefficiency, it is preferred that all components of the functional pgloperon are regulated and expressed simultaneously.

It is important to realize that only the functional oligosaccharyltransferase (OTase) should originate from Campylobacter spp., preferablyC. jejuni, and that the one or more specific glycosyltransferasescapable of assembling an oligosaccharide on a lipid carrier mayoriginate from the host cell or be introduced recombinantly into saidhost cell, the only functional limitation being that the oligosaccharideassembled by said glycosyltransferases can be transferred from the lipidcarrier to the target protein having one or more optimized consensussequences by the OTase. Hence, the selection of the host cell comprisingspecific glycosyltransferases naturally and/or incapacitating specificglycosyltransferases naturally present in said host as well as theintroduction of heterologous specific glycosyltransferases will enablethose skilled in the art to vary the N-glycans bound to the optimizedN-glycosylation consensus site in the proteins of the present invention.

As a result of the above, the present invention provides for theindividual design of N-glycan-patterns on the proteins of the presentinvention. The proteins can therefore be individualized in theirN-glycan pattern to suit biological, pharmaceutical and purificationneeds.

In a preferred embodiment, the proteins of the present invention maycomprise one but also more than one, preferably at least two, preferablyat least 3, more preferably at least 5 of said N-glycosylated optimizedamino acid sequences.

The presence of one or more N-glycosylated optimized amino acidsequence(s) in the proteins of the present invention can be of advantagefor increasing their antigenicity, increasing their stability, affectingtheir biological activity, prolonging their biological half-life and/orsimplifying their purification.

The optimized consensus sequence may include any amino acid exceptproline in position(s) X and Z. The term “any amino acids” is meant toencompass common and rare natural amino acids as well as synthetic aminoacid derivatives and analogs that will still allow the optimizedconsensus sequence to be N-glycosylated by the OTase. Naturallyoccurring common and rare amino acids are preferred for X and Z. X and Zmay be the same or different.

It is noted that X and Z may differ for each optimized consensussequence in a protein according to the present invention.

The N-glycan bound to the optimized consensus sequence will bedetermined by the specific glycosyltransferases and their interactionwhen assembling the oligosaccharide on a lipid carrier for transfer bythe OTase. Those skilled in the art can design the N-glycan by varyingthe type(s) and amount of the specific glycosyltransferases present inthe desired host cell.

N-glycans are defined herein as monO-, oligO- or polysaccharides ofvariable compositions that are linked to an ε-amide nitrogen of anasparagine residue in a protein via an N-glycosidic linkage. Preferably,the N-glycans transferred by the OTase are assembled on anundecaprenol-pyrophosphate lipid-anchor that is present in thecytoplasmic membrane of gram-negative or positive bacteria. They areinvolved in the synthesis of O antigen, O polysaccharide andpeptidoglycan (Bugg, T. D., and Brandish, P. E. (1994). Frompeptidoglycan to glycoproteins: common features of lipid-linkedoligosaccharide biosynthesis. FEMS Microbiol Lett 119, 255-262; Valvano,M. A. (2003). Export of O-specific lipopolysaccharide. Front Biosci 8,s452-471).

In a preferred embodiment, the recombinant protein of the presentinvention comprises one or more N-glycans selected from the group ofN-glycans from Campylobacter spp., preferably C. jejuni, the N-glycansderived from oligO- and polysaccharides transferred to O antigen formingO polysaccharide in Gram-negative bacteria or capsular polysaccharidesfrom Gram-positive bacteria, preferably: P. aeruginosa 09, 011; E. coli07, 09, 016, 0157 and Shigella dysenteriae O1 and engineered variantsthereof obtained by inserting or deleting glycosyltransferases andepimerases affecting the polysaccharide structure.

In a further preferred embodiment, the recombinant protein of thepresent invention comprises two or more different N-glycans.

For example, different N-glycans on the same protein can prepared bycontrolling the timing of the expression of specificglycosyltransferases using early or late promoters or introducingfactors for starting, silencing, enhancing and/or reducing the promoteractivity of individual specific glycosyltransferases. Suitable promotersand factors governing their activity are routinely available to those inthe art.

There is no limitation on the origin of the recombinant protein of theinvention. Preferably said protein is derived from mammalian, bacterial,viral, fungal or plant proteins. More preferably, the protein is derivedfrom mammalian, most preferably human proteins. For preparing antigenicrecombinant proteins according to the invention, preferably for use asactive components in vaccines, it is preferred that the recombinantprotein is derived from a bacterial, viral or fungal protein.

In a further preferred embodiment, the present invention provides forrecombinant proteins wherein either the protein and/or the N-glycan(s)is (are) therapeutically and/or prophylactically active. Theintroduction of at least one optimized and N-glycosylated consensussequence can modify or even introduce therapeutic and/or prophylacticactivity in a protein. In a more preferred embodiment, it is the proteinand/or the N-glycan(s) that is (are) immunogenically active. In thiscase, the introduced N-glycosylation(s) may have a modifying effect onthe proteins biological activity and/or introduce new antigenic sitesand/or may mask the protein to evade degrading steps and/or increase thehalf-life.

The recombinant proteins of the present invention can be efficientlytargeted to the outer membrane and/or surface of host cells, preferablybacteria, more preferably gram-negative bacteria. For assisting thesurface display and/or outer membrane localization, it is preferred thatthe recombinant protein of the invention further comprise at least onepolypeptide sequence capable of targeting said recombinant protein tothe outer membrane and/or cell surface of a bacterium, preferably agram-negative bacterium.

In a preferred embodiment, the recombinant protein of the invention isone, wherein said targeting polypeptide sequence is selected from thegroup consisting of type II signal peptides (Paetzel, M., Karla, A.,Strynadka, N. C., and Dalbey, R. E. 2002. Signal peptidases. Chem Rev102: 4549-4580.) or outer membrane proteins (reviewed in Wemerus, H.,and Stahl, S. 2004. Biotechnological applications for surface-engineeredbacteria. Biotechnol Appl Biochem 40: 209-228.), preferably selectedfrom the group consisting of the full length protein or the signalpeptides of OmpH1 from C. jejuni, JIpA from C. jejuni, outer membraneproteins from E. coli, preferably OmpS, OmpC, OmpA, OprF, PhoE, LamB,Lpp′OmpA (a fusion protein for surface display technology, seeFrancisco, JA₁ Earhart, C. F., and Georgiou, G. 1992. Transport andanchoring of beta-lactamase to the external surface of Escherichia coli.Proc Natl Acad Sci USA 89: 2713-2717.), and the lnp protein fromPseudomonas aeruginosa.

In a different aspect, the present invention relates to a nucleic acidencoding a recombinant protein according to the invention. Preferably,said nucleic acid is a mRNA, a DNA or a PNA, more preferably a mRNA or aDNA, most preferably a DNA. The nucleic acid may comprise the sequencecoding for said protein and, in addition, other sequences such asregulatory sequences, e.g. promoters, enhancers, stop codons, startcodons and genes required to regulate the expression of the recombinantprotein via the mentioned regulatory sequences, etc. The term “nucleicacid encoding a recombinant protein according to the invention” isdirected to a nucleic acid comprising said coding sequence andoptionally any further nucleic acid sequences regardless of the sequenceinformation as long as the nucleic acid is capable of producing therecombinant protein of the invention in a host cell containing afunctional pgl operon from Campylobacter spp., preferably C. jejuni.More preferably, the present invention provides isolated and purifiednucleic acids operably linked to a promoter, preferably linked to apromoter selected from the group consisting of known inducible andconstitutive prokaryotic promoters, more preferably the tetracyclinepromoter, the arabinose promoter, the salicylate promoter, lac-, trc-,and lac promotors (Baneyx, F. (1999). Recombinant protein expression inEscherichia coli. Curr Opin Biotechnol 10, 411-421; Billman-Jacobe, H.(1996). Expression in bacteria other than Escherichia coli. Curr OpinBiotechnol 7, 500-504.). Said operably linked nucleic acids can be usedfor, e.g. vaccination.

Furthermore, another aspect of the present invention relates to a hostcell comprising a nucleic acid and/or a vector according to the presentinvention. The type of host cell is not limiting as long as itaccommodates a functional pgl operon from C. jejuni and one or morenucleic acids coding for recombinant target protein(s) of the presentinvention. Preferred host cells are prokaryotic host cells, morepreferably bacteria, most preferably those selected from the groupconsisting of Escherichia ssp., Campylobacter ssp., Salmonella ssp.,Shigella ssp., Helicobacter ssp., Pseudomonas ssp., Bacillus ssp.,preferably Escherichia coli, more preferably E. coli strains Top10,W3110, CLM24, BL21, SCM6 and SCM7 (Feldman et al., (2005). EngineeringN-linked protein glycosylation with diverse O antigen lipopolysaccharidestructures in Escherichia coli. Proc. Natl. Acad. Sci. USA 102,3016-3021; Alaimo, C, Catrein, I., Morf, L., Marolda, C. L., Callewaert,N., Valvano, M. A., Feldman, M. F., Aebi, M. (2006). Two distinct butinterchangeable mechanisms for flipping of lipid-linkedoligosaccharides. EMBO Journal 25, 967-976) and S. enterica strainsSL3261 (Salmonella enterica sv. Typhimurium LT2 (delta) aroA, seeHoiseth, S. K., and Stocker, B. A. 1981, Aromatic-dependent Salmonellatyphimurium are non-virulent and effective as live vaccines. Nature291:238-239), SL3749 (Salmonella enterica sv. Typhimurium LT2 waaL, seeKaniuk et al., J. Biol. Chem. 279: 36470-36480) and SL3261 ΔwaaL.

In a more preferred embodiment, the host cell according to the inventionis one that is useful for the targeting to the outer membrane and/orsurface display of recombinant proteins according to the invention,preferably one, wherein said host cell is a recombinant gram-negativebacterium having:

-   -   i) a genotype comprising nucleotide sequences encoding for        -   a) at least one natural or recombinant specific            glycosyltransferase for the assembly of an oligosaccharide            on a lipid carrier,        -   b) at least one natural or recombinant prokaryotic            oligosaccharyl transferase (OTase) from Campylobacter spp.,            preferably C. jejuni,        -   c) at least one recombinant protein according to the            invention, preferably a protein further comprising a            targeting polypeptide, and    -   ii) a phenotype comprising a recombinant N-glycosylated protein        according to the invention that is located in and/or on the        outer membrane of the gram-negative bacterium.

The host cell for the above embodiment is preferably selected from thegroup consisting of Escherichia ssp., Campylobacter ssp., Shigella ssp,Helicobacter ssp. and Pseudomonas ssp., Salmonella ssp., preferably E.coli, more preferably E. coli strains Top10, W3110, CLM24, BL21, SCM6and SCM7, and S. enterica strains SL3261, SL3749 and SL326iδwaaL (seeHoiseth, S. K., and Stocker, B. A. 1981. Aromatic-dependent Salmonellatyphimurium are non-virulent and effective as live vaccines. Nature 291:238-239), SL3749 (Kaniuk, N. A., Vinogradov, E., and Whitfield, C. 2004.Investigation of the structural requirements in the lipopolysaccharidecore acceptor for ligation of O antigens in the genus Salmonella: WaaL“ligase” is not the sole determinant of acceptor specificity. J BiolChem 279: 36470-36480).

Because preferred proteins of the present invention may have atherapeutic or prophylactic activity by themselves and/or due to theintroduced N-glycosylation sites, they can be used for the preparationof a medicament. The type of protein for practicing the invention is notlimited and, therefore, proteins of the invention such as EPO,IFN-alpha, TNFalpha, IgG, IgM, IgA, interleukins, cytokines, viral andbacterial proteins for vaccination like C. jejuni proteins such as HisJ(CjO734c), AcrA (CjO367c), OmpH1 (CjO982c), Diphteria toxin (CRM 197),Cholera toxin, P. aeruginosa exoprotein, to name just a few, and havingintroduced therein the optimized N-glycosylated consensus sequence areuseful for preparing a medicament (Wyszynska, A., Raczko, A., Lis, M.,and Jagusztyn-Krynicka, E. K. (2004). Oral immunization of chickens withavirulent Salmonella vaccine strain carrying C. jejuni 72Dz/92 cjaA geneelicits specific humoral immune response associated with protectionagainst challenge with wild-type Campylobacter. Vaccine 22, 1379-1389).

In addition, the nucleic acids and/or vectors according to the inventionare also useful for the preparation of a medicament, preferably for usein gene therapy.

Moreover, a host cell according to the invention, preferably one thathas a phenotype comprising an N-glycosylated recombinant protein of theinvention that is located in and/or on the outer membrane of abacterium, preferably a gram-negative bacterium, more preferably one ofthe above-listed gram-negative bacteria, is particularly useful for thepreparation of a medicament.

More preferably, a protein of the invention is used for the preparationof a medicament for the therapeutic and/or prophylactic vaccination of asubject in need thereof.

In a more preferred embodiment, the present invention relates to the useof a nucleic acid and/or a vector according to the invention for thepreparation of a medicament for the therapeutic and/or prophylacticvaccination of a subject in need thereof, preferably by gene therapy.

The host cells of the invention displaying said N-glycosylatedrecombinant proteins are particularly useful for preparing vaccines,because the displayed N-glycosylated proteins are abundantly present onthe host cell's surface and well accessible by immune cells, inparticular their hydrophilic N-glycans, and because the host cells havethe added effect of an adjuvant, that, if alive, may even replicate tosome extent and amplify its vaccination effects.

Preferably, the host cell for practicing the medical aspects of thisinvention is an attenuated or killed host cell.

Another advantage of the use of the inventive host cells for preparingmedicaments, preferably vaccines, is that they induce IgA antibodies dueto the cellular component.

Preferably, said host cells are used according to the invention forinducing IgA antibodies in an animal, preferably a mammal, a rodent,ovine, equine, canine, bovine or a human. It is preferred that saidsubject in need of vaccination is avian, mammalian or fish, preferablymammalian, more preferably a mammal selected from the group consistingof cattle, sheep, equines, dogs, cats, and humans, most preferablyhumans. Fowls are also preferred.

A further aspect of the present invention relates to a pharmaceuticalcomposition, comprising at least one protein, at least one nucleic acid,a least one vector and/or at least one host cell according to theinvention. The preparation of medicaments comprising proteins or hostcells, preferably attenuated or killed host cells, and the preparationof medicaments comprising nucleic acids and/or vectors for gene therapyare well known in the art. The preparation scheme for the finalpharmaceutical composition and the mode and details of itsadministration will depend on the protein, the host cell, the nucleicacid and/or the vector employed.

In a preferred embodiment, the pharmaceutical composition of theinvention comprises a pharmaceutically acceptable excipient, diluentand/or adjuvant.

The present invention provides for a pharmaceutical compositioncomprising at least one of the following, (i) a recombinant protein, ahost cell, a nucleic acid and/or a recombinant vectorbeing/encoding/expressing a recombinant protein according to the presentinvention, and (ii) a pharmaceutically acceptable excipient, diluentand/or adjuvant.

Suitable excipients, diluents and/or adjuvants are well-known in theart. An excipient or diluent may be a solid, semi-solid or liquidmaterial which may serve as a vehicle or medium for the activeingredient. One of ordinary skill in the art in the field of preparingcompositions can readily select the proper form and mode ofadministration depending upon the particular characteristics of theproduct selected, the disease or condition to be treated, the stage ofthe disease or condition, and other relevant circumstances (Remington'sPharmaceutical Sciences, Mack Publishing Co. (1990)). The proportion andnature of the pharmaceutically acceptable diluent or excipient aredetermined by the solubility and chemical properties of thepharmaceutically active compound selected, the chosen route ofadministration, and standard pharmaceutical practice. The pharmaceuticalpreparation may be adapted for oral, parenteral or topical use and maybe administered to the patient in the form of tablets, capsules,suppositories, solution, suspensions, or the like. The pharmaceuticallyactive compounds of the present invention, while effective themselves,can be formulated and administered in the form of their pharmaceuticallyacceptable salts, such as acid addition salts or base addition salts,for purposes of stability, convenience of crystallization, increasedsolubility, and the like.

A further aspect of the present invention is directed to a method forproducing N-linked glycosylated proteins, comprising the steps of:

-   -   a) providing a recombinant organism, preferably a prokaryotic        organism, comprising nucleic acids coding for        -   i) a functional pgl operon from Campylobacter spp.,            preferably C. jejuni, and        -   ii) at least one recombinant target protein comprising one            or more of the following N-glycosylated optimized amino acid            consensus sequence(s):            D/E-X-N-Z-S/T,    -   wherein X and Z may be any natural amino acid except Pro, and        wherein at least one of said N-glycosylated optimized amino acid        consensus sequence(s) is introduced, and    -   b) culturing the recombinant organism in a manner suitable for        the production and N-glycosylation of the target protein(s).

Preferably, the target protein is one of the above described recombinantproteins according to the invention.

In a preferred method of the invention, the functional pgl operon fromCampylobacter spp., preferably C. jejuni, comprises nucleic acids codingfor

-   -   i) recombinant OTase from Campylobacter spp., preferably C.        jejuni, and    -   ii) recombinant and/or natural specific glycosyltransferases        from Campylobacter spp., preferably C. jejuni, and/or    -   iii) recombinant and/or natural specific glycosyltransferases        from species other than Campylobacter spp.,        for the assembly of an oligosaccharide on a lipid carrier to be        transferred to the target protein by the OTase.

Moreover, in a preferred embodiment the present invention relates to amethod for preparing a host cell according to the invention comprisingthe steps of:

-   -   i) providing a gram-negative bacterium,    -   ii) introducing into said bacterium at least one nucleotide        sequence encoding for        -   a) at least one recombinant specific glycosyltransferase for            the assembly of an oligosaccharide on a lipid carrier,            and/or        -   b) at least one recombinant oligosaccharyl transferase            (OTase) from Campylobacter spp., preferably C. jejuni,            and/or        -   c) at least one recombinant protein comprising one or more            of the following N-glycosylated optimized amino acid            consensus sequence(s):            D/E-X-N-Z-S/T,    -   wherein X and Z may be any natural amino acid except Pro, and        wherein at least one of said N-glycosylated optimized amino acid        consensus sequence(s) is introduced, and    -   iii) culturing said bacterium until at least one recombinant        N-glycosylated protein coded by the nucleotide sequence of c) is        located in and/or on the outer membrane of the gram-negative        bacterium.

For practicing the preferred methods above, the recombinant procaryoticorganism or host cell is preferably selected from the group of bacteriaconsisting of Escherichia ssp., Campylobacter ssp., Salmonella ssp.,Shigella ssp., Helicobacter ssp., Pseudomonas ssp., Bacillus ssp.,preferably Escherichia coli, preferably E. coli strains Top 10, W3110,W3110ΔwaaL, BL21, SCM6 and SCM7, and S. enterica strains SL3261, SL3749and SL3261 ΔwaaL.

Another preferred method for producing, isolating and/or purifying arecombinant protein according to the invention comprises the steps of:

-   -   a) culturing a host cell,    -   b) removing the outer membrane of said recombinant gram-negative        bacterium; and    -   c) recovering said recombinant protein.

Exemplary methods for removing the outer membrane of a cell, preferablya prokaryotic cell, more preferably a gram-negative bacterial cell, aresuitable enzymatic treatment methods, osmotic shock detergentsolubilisation and the French press method.

Most preferred, the present invention relates to a method, whereinrecombinant or natural specific glycosyltransferases from species otherthan Campylobacter spp., preferably C. jejuni, are selected from thegroup of glycosyltransferases and epimerases originating from bacteria,archea, and/or eukaryota that can be functionally expressed in said hostcell.

Bioconjugate Vaccines

An embodiment of the invention involves novel bioconjugate vaccines. Afurther embodiment of the invention involves a novel approach forproducing such bioconjugate vaccines that uses recombinant bacterialcells that directly produce immunogenic or antigenic bioconjugates. Inone embodiment, bioconjugate vaccines can be used to treat or preventbacterial diseases, such as diarrhea, nosocomial infections andmeningitis. In further embodiments, biooconjugate vaccines may havetherapeutic and/or prophylactic potential for cancer or other diseases.

Conjugate vaccines can be administered to children to protect againstbacterial infections and can provide a long lasting immune response toadults. Constructs of the invention have been found to generate an IgGresponse in animals. It has been found that an IgG response to aShigella O-specific polysaccharide-protein conjugate vaccine in humanscorrellates with immune protection in humans. (Passwell, J. H. et al.,“Safety and Immunogenicity of Improved Shigella O-SpecificPolysaccharide-Protein Conjugate Vaccines in Adults in Israel” Infectionand Immunity, 69(3):1351-1357 (March 2001).) It is believed that thepolysaccharide (i.e. sugar residue) triggers a short-term immuneresponse that is sugar-specific. Indeed, the human immune systemgenerates a strong response to specific polysaccharide surfacestructures of bacteria, such as O-antigens and capular polysaccharides.However, since the immune response to polysaccharides is IgM dependent,the immune system develops no memory. The protein carrier that carriesthe polysaccharide triggers an IgG response that is T-cell dependent andthat provides long lasting protection since the immune system developsmemory.

A typical vaccination dosage for humans is about 1 to 25 μg, preferablyabout 1 μg to about 10 μg, most preferably about 10 μg. Optionally, avaccine, such as a bioconjugate vaccine of the present invention,includes an adjuvant.

Synthesized complexes of polysaccharides (i.e., sugar residues) andproteins (i.e., protein carriers) can be used as conjugate vaccines toprotect against a number of bacterial infections. In one aspect, theinstant invention is directed to a novel bioengineering approach forproducing immunogenic conjugate vaccines that provide advantages overclassical chemical conjugation methods. In one embodiment, the approachinvolves in vivo production of glycoproteins in bacterial cells, forexample, Gram-negative cells such as E. coli.

The biosynthesis of different polysaccharides is conserved in bacterialcells. The polysaccharides are assembled on carrier lipids from commonprecursors (activated sugar nucleotides) at the cytoplasmic membrane bydifferent glycosyltransferases with defined specificity.Lipopolysaccharides (LPS) are provided in gram-negative bacteria only,e.g. Shigella spp., Pseudomonas spp. and E. coli (ExPEC, EHEC).

The synthesis of lipopolysaccharides (LPS) starts with the addition of amonosaccharide to the carrier lipid undecaprenyl phosphate at thecytoplasmic side of the membrane. The antigen is built up by sequentialaddition of monosaccharides from activated sugar nucleotides bydifferent glycosyltransferases and the lipid-linked polysaccharide isflipped through the membrane by a flippase. The antigen-repeating unitis polymerized by an enzymatic reaction. The polysaccharide is thentransferred to the Lipid A by the Ligase WaaL forming the LPS that isexported to the surface, whereas the capsular polysaccharide is releasedfrom the carrier lipid after polymerization and exported to the surface.The biosynthetic pathway of these polysaccharides enables the productionof LPS bioconjugates in vivo, capturing the polysaccharides in theperiplasm to a protein carrier. Bioconjugates, such as LPSbioconjugates, are preferred in the present invention.

As shown in FIG. 5A, both Gram-positive and Gram-negative bacteria havea cell membrane that is surrounded by capsular polysaccharides.Gram-negative bacteria additionally have an outer membrane over the cellmembrane, with a periplasmic space separating the two membranes. Inaddition, they contain lipopolysacharides at the surface. WhenGram-negative bacteria, such as E. coli, is used to produce a conjugatevaccine of the present invention, the glycoprotein used in the conjugatevaccine is assembled in the periplasmic space.

Conjugate vaccines have been successfully used to protect againstbacterial infections. The conjugation of an antigenic polysaccharide toa protein carrier is required for protective memory response, aspolysaccharides are T-cell independent antigens. Polysaccharides havebeen conjugated to protein carriers by different chemical methods, usingactivation reactive groups in the polysaccharide as well as the proteincarrier.

FIG. 6A shows the production process of conjugate vaccines usingtechnology of the invention compared to the currently used process.Currently, conjugate vaccines are produced using two fermentation runsand after several purification and chemical cleavage steps, asschematically shown in the top panel. This current approach has a numberof problems. First, large scale cultivation of pathogenic organisms isrequired. Second, the conjugation approach is dependent on thepolysaccharide. Third, the approach has low reproducibility. Fourth, theapproach has low homogeneity due to unspecific conjugation. Fifth, theapproach also has low purity due to excess of polysaccharide inconjugation. Finally, the current approach provides yields of less than20%.

As shown in the bottom panel of FIG. 6A, in an embodiment, theinnovative technology of the invention can be used to develop conjugatevaccines (e.g., bioconjugate vaccines) completely in vivo withnon-pathogenic cells, avoiding chemical reactions and providing highpurity after a few purification steps. This novel method also allows forthe production of bioconjugate vaccines that are not feasible usingcurrent methods. Moreover, the conjugation and purification process isindependent of the polysaccharide antigen that is used. As a result,bioconjugate vaccines can be engineered faster using novel glycanstructures. The increased homogeneity of resulting conjugates and theimproved reproducibility (i.e., no batch to batch variability) of suchconjugates makes this a highly attractive process from quality controland regulatory perspectives. In addition, the novel method provides goodyield (30-60 mg/L and up to 200 mg/L).

The present invention is directed to a novel conjugation processinvolving engineering bacterial cells to produce the final bioconjugatevaccines. One embodiment of the invention allows the production ofbioconjugate vaccines in vivo, circumventing the chemical conjugationand therefore simplifying the production process. The technologyincludes a novel genetic/enzymatic mechanism for the in vivo synthesisof novel bioconjugates consisting of protein-linked saccharides.

The basis of one aspect of the invention includes the discovery thatCampylobacter jejuni contains a general N-linked protein glycosylationsystem, an unusual feature for prokaryotic organisms. Various proteinsof C. jejuni have been shown to be modified by a heptasaccharide. Thisheptasaccharide is assembled on undecaprenyl pyrophosphate, the carrierlipid, at the cytoplasmic side of the inner membrane by the stepwiseaddition of nucleotide activated monosaccharides catalyzed by specificglycosyltransferases. The lipid-linked oligosaccharide then flip-flops(diffuses transversely) into the periplasmic space by a flipppase, e.g.,PgIK. In the final step of N-linked protein glycosylation, theoligosaccharyltransferase (e.g., PgIB) catalyzes the transfer of theoligosaccharide from the carrier lipid to Asn residues within theconsensus sequence Asp/Glu-Xaa-Asn-Zaa-Ser/Thr (i.e., D/E-X-N-Z-S/T),where the Xaa and Zaa can be any amino acid except Pro (FIG. 7A). Wehave successfully transferred the glycosylation cluster for theheptasaccharide into E. coli and were able to produce N-linkedglycoproteins of Campylobacter.

We have been able to demonstrate that PgIB does not have a strictspecificity for the lipid-linked sugar substrate. The antigenicpolysaccharides assembled on undecaprenyl pyrophosphate are captured byPglB in the periplasm and transferred to a protein carrier (Feldman,2005; Wacker, M., et al., Substrate specificity of bacterialoligosaccharyltransferase suggests a common transfer mechanism for thebacterial and eukaryotic systems. Proc Natl Acad Sci USA, 2006. 103(18):p. 7088-93.) The enzyme will also transfer a diverse array ofundecaprenyl pyrophosphate (UPP) linked oligosaccharides if they containan N-acetylated hexosamine at the reducing terminus. The nucleotidesequence for pgIB is provided at SEQ. ID NO. 1, whereas the amino acidsequence for PgIB is provided at SEQ. ID. NO. 2.

FIGS. 7A and 7B show schematics of the protein glycosylation pathway(i.e., N-glycosylation system) of the present invention. In anembodiment, the protein glycosylation pathway of C. jejuni (e.g.,including pgl operon) can be introduced into E. coli. In FIG. 7A, anoligosaccharide, specifically a heptasaccharide made of fiveN-acetyl-D-galactosamine units, one glucose unit and one2,4-diacetamidO-2,4,6-trideoxy-D-glucose unit, is assembled onto a lipidcarrier, undecaprenylpyrophosphate (UDP), using glycosyltransferases(e.g., pglA, pglC, pglH, J; I) at the cytoplasmic side of the innermembrane and is transferred to the periplasmic space by way of aflippase called PgIK. Separately, a carrier protein depicted as a spiraland containing consensus sequence D/E-X-N-Z-S/T (i.e.,Asp/Glu-Xaa-Asn-Zaa-Ser/Thr) is translated in the cytoplasm and issecreted into the periplasmic space. In the final step, anoligosaccharyl transferase (OST or OTase) (e.g., PgIB) transfers theheptasaccharide to Asn residues within a consensus sequence of thecarrier protein to produce a glycoprotein.

FIG. 7B also shows biosynthesis of a polysaccharide (i.e., an antigenicpolysaccharide or antigen) by stepwise action of glycosyltransferases,and transfer of the O-antigen to the periplasm by way of flippase,followed by polymerization into a polysaccharide using a polymerase(e.g., wzy). Separately, a carrier protein, such as EPA, is produced andsecreted into the periplasm. An oligosaccharyl transferase (OST orOTase), such as PgIB, has relaxed substrate specificity and transfersthe polysaccharide from a lipid carrier to Asn in the consensus sequencewithin EPA.

FIG. 8A shows a schematic depicting an embodiment of the expressionplatform for bioconjugate production of the present invention. Thetechnology of the invention is versatile in that various existingcarrier proteins can be employed, so long as the carrier proteincontains or is modified to contain the consensus sequence, as discussedearlier. In particular, FIG. 8A illustrates the construction of anexpression host, such as an engineered E. coli bacterium in anembodiment of the invention. Such an E. coli contains the generalcomponents of a glycosylation system (i.e., an OST/OTase, e.g., PgIB,and a protein carrier, e.g. EPA). Such components can be integrated intothe genome of an E. coli strain. In addition, the Ligase WaaL as well asWecG are deleted. Additionally, specific components for polysaccharideantigen expression (i.e., a polysaccharide synthesis gene clustercontaining, for example, glycosyl transferase, polymerase, flippase, andsugar biosynthesis enzymes) can be provided by the addition of anexchangeable plasmid. This construction allows for specificglycosylation of the protein carrier with a polysaccharide of choice invivo.

In an embodiment of the expression system for a bacterial bioconjugatethat is compatible with Good Manufacturing Practices (GMP), DNA encodingthe inducible oligosaccharyltransferase and carrier protein can bestably integrated into a bacterial (e.g., E. coli) genome such thatgenes for antibiotic selection can be omitted. For example, as shown inFIG. 5B, PgIB and EPA is integrated into genomic DNA, whereas plasmidDNA, which is interchangeable (i.e., exchangeable), encodes apolysaccharide synthesis gene cluster.

In another embodiment, FIG. 8B shows an expression system for abacterial bioconjugate that includes three plasmids. A first plasmidcodes for the carrier protein, e.g., AcrA from Campylobacter jejuni,which has two N-glycosylation sites and is directed to the periplasm bya PelB signal peptide. A second plasmid codes for the OST/OTase, e.g.,PgIB from C. jejuni, which is membrane-bound. A third plasmid is anative plasmid that codes, e.g., for a polysaccharide (O antigen)synthesis cluster, such as that for Shigella dysenteriae O1.

In an embodiment, an expression plasmid for a bacterial O antigen, suchas the Shigella dysenteriae O1 antigen, can be constructed as in pGVXN64shown in FIG. 6B. This plasmid encodes all enzymes necessary tosynthesize the polysaccharides in the Shigella dysenteriae strain thatmake up the O1 serotype. These enzymes are listed in the left-handcolumn of FIG. 6B. Vector pGVXN64 expressing the Shigella dysenteriae O1antigen was constructed by digestion of pLARFR1 (Vanbleu, E. et al.,“Genetic and physical map of the pLAFR1 vector” DNA Seq. 15(3):225-227(2004)) with EcoR1 followed by insertion of an oligonucleotide cassette(5′-AATTCTGCAGGATCCTCTAGAAGCTTGG (SEQ. ID NO. 3) and5′-AATTCCAAGCTTCTAGAGGATCCTGCAG (SEQ. ID NO. 4). The BamH1 fragment ofpSDM7 (Falt, I. et al., “Construction of recombinant aroA salmonellaestably producing the Shigella Dysenteriae serotype 1 O-antigen andstructural characterization of the Salmonella/Shigella hybrid LPS”Microb. Pathog. 20(1):11-30 (1996)) containing the rfb and rfp clusterof Shigella dysenteriae O1 was then cloned via the BamH1 site into theoligonucleotide cassette containing pLAFR1. The complete nucleotidesequence encoding the Shigella dysenteriae O1 antigen in the pGVXN64plasmid is set forth as SEQ. ID NO.: 5 in the Sequence Listing providedbelow.

The host organism for an expression system of the invention can be,e.g., an Escherichia coli strain such as Escherichia coli W31110ΔwaaL.The deletion of WaaL prevents the transfer of any polysaccharide to thelipid A core. The chromosomal copy of WaaL can also be replaced by PglB.The strain also contains mutation in wbbL, therefore it does not produceany E. coli O16 polysaccharide. To further increase the production ofcarrier lipid linked polysaccharide, wecG has been deleted to preventthe formation of ECA (Entero Common Antigen).

In one aspect, the instant invention is further directed to thedevelopment of bioconjugate vaccines, preferably LPS bioconjugatevaccines, against one or more Shigella species, which are invasive,gram-negative bacteria. Shigella species cause Shigellosis, a severeinflammation of the colon. There are 165 million cases in the worldevery year, with 70% of such cases being in children under 5 years ofage. In developing countries, Shigellosis causes 1.1 million of deathsper year. This is a serious disease that is spread via the fecal-oralroute and is highly transmissible. Potential groups that would benefitfrom immunization against Shigella species include, for example,children, travelers and people in refugee camps.

There are four different serogroups of Shigella, namely, S. dysenteriae,S. flexneri, S. sonnei and S. boydii. In embodiments of the presentinvention, immunogenic bioconjugates can be made against each of thesedifferent serogroups of Shigella. For example, FIG. 14B providesdifferent serotypes of Shigella and the polysaccharide structure thatdefines their antigenicity (i.e., Shigella O-antigens).

In further embodiments of the present invention, immunogenic LPSbioconjugates could be made against other bacteria using the teachingsin this specification, including bacteria: (1) that cause nosocomialinfections, such as Pseudomonas aeruginosa; and (2) that cause urinarytract infection, such as Extraintestinal E. coli (ExPEC).

In an embodiment, the inventors have developed a Shigella dysenteriae O1LPS bioconjugate vaccine (also referred to as a S. dysenteriaebioconjugate), using genetically engineered E. coli with simplefermentation and purification methods. FIG. 9 shows production ofShigella bioconjugates. The top panel shows the synthesis ofbioconjugates in E. coli. In an embodiment, the O-antigen repeating unitof S. dysenteriae O1 is assembled on the carrier lipid undecaprenylpyrophosphate (UPP), flipped to the periplasmic space and polymerized.The structure of S. dysenteriae O1 is as follows and is also provided inthe middle right of FIG. 9:

PgIB transfers the activated polysaccharide to Asn residues of proteincarriers, forming the Shigella bioconjugates. The protein carrier canbe, for example, AcrA or a protein carrier that has been modified tocontain the consensus sequence for protein glycosylation, i.e.,D/E-X-N-Z-S/T, wherein X and Z can be any amino acid except proline(e.g., a modified Exotoxin Pseudomonas aeruginosa (EPA)). EPA has beenused successfully in conjugate vaccines.

In an embodiment illustrated in FIG. 9, periplasmic proteins of E. colicells expressing the modified EPA in the presence of PgIB and the O1polysaccharide cluster were separated by SDS page and, after transfer tonitrocellulose, EPA was immunodetected with an antiserum that was raisedagainst EPA (lane 2). In lane 1, periplasmic proteins of E. coli cellsexpressing the Campylobacter protein AcrA in the presence of PgIB andthe O1 polysaccharide cluster were separated and immunodetected with anantiserum that was raised against AcrA. Both proteins were glycosylatedwith the O1-polysaccharide cluster. In the lowest panel of FIG. 9, theLPS from E. coli were separated by SDS-PAGE and visualized by SilverStaining. The left lane depicts the LPS extracted from a strain notexpressing the WaaL, whereas the right lane shows the typical O1 LPSpattern. Both strains are expressing the polysaccharide biosynthesiscluster of S. dysenteriae O1.

The production of a bacterial bioconjugate, such as a Shigellabioconjugate, is described in an embodiment in further detail withreference to FIGS. 10A and 10B. Prior to assembly of the bacterialbioconjugate using a bacterial system, such as E. coli, it is necessaryto introduce into the bacterial system certain genetic sequences codingfor the various enzymes and proteins to be used, as discussed earlierwith reference to FIGS. 8A and 8B. For example, this includes anOST/Otase, preferably from C. jejuni (e.g., PglB), a protein carrier(e.g. EPA) and a gene cluster directed to antigenic polysaccharidesynthesis (e.g., the gene cluster for S. dysenteriae O1 polysaccharidesynthesis).

FIG. 10A shows Step 1 in the development of a bacterial bioconjugate,namely, the biosynthesis of a polysaccharide, such as the O-antigen ofS. dysenteriae Serotype O1. In this step, the antigen is synthesized onthe carrier lipid undecaprenyl pyrophosphate (UPP), and then transferredinto the periplasm using a flippase. The antigen is polymerized by thepolymerase Wzy and transferred to the lipid A core by the ligase WaaL.To transfer the polysaccharide to a protein carrier, the ligase isreplaced by the oligosaccharyltransferase; PglB.

Step 2 in the production of a bacterial bioconjugate involvesengineering a suitable protein carrier. Protein carriers that are usefulpreferably should have certain immunological and pharmacologicalfeatures. From an immunological perspective, preferably, a proteincarrier should: (1) have T-cell epitopes; (2) be capable of deliveringan antigen to antigen presenting cells (APCs) in the immune system; (3)be potent and durable; and (4) be capable of generating anantigen-specific systemic IgG response. From a pharmacologicalperspective, preferably, a protein carrier should: (1) be non-toxic; and(2) be capable of delivering antigens efficiently across intactepithelial barriers. More preferably, in addition to these immunologicaland pharmacological features, a protein carrier suitable for theproduction of a bacterial bioconjugate should: (1) be easily secretedinto the periplasmic space; and (2) be capable having antigen epitopesreadily introduced as loops or linear sequences into it.

The inventors have found genetically detoxified Pseudomonas aeruginosaExotoxin (EPA) and the Campylobacter protein AcrA to be suitable proteincarriers, most preferably EPA. AcrA contains natural glycosylation siteswhereas EPA needs to be modified to encode glycosylation sites.Preferably, EPA is modified to introduce two glycosylation sitesdirected to the Shigella O1 antigen. More preferably, two consensussequences are introduced as discussed in Example 10.

The amino acid sequence of EPA, as modified in an embodiment of thisinvention to contain two glycosylation sites, is provided as SEQ. IDNO.: 6 (with signal sequence) and SEQ. ID NO.: 7 (without signalsequence) in the Sequence Listing provided below. The glycosylationsites in each of SEQ. ID NO.: 6 and SEQ. ID NO.: 7 are denoted with anunderline.

FIG. 10B shows a schematic of a carrier protein, such as EPA onto whichN-glycosylation sites can be designed as Step 3 in the production of abacterial bioconjugate. N-glycosylation sites require introduction ofthe consensus sequences discussed previously, namely, insertion ofD/E-X-N-Z-S/T sequons, wherein X and Z may be any natural amino acidexcept proline. We have found that such consensus sequences preferablyare introduced through surface loops, by insertion rather than mutationand considering using flanking residues to optimize the operation of theN-glycosylation site.

FIG. 11 shows bicoonjugates that elicit an immune response againstShigella O1 polysaccharide in mice. O1-AcrA and O1-EPA was purified byaffinity column and anionic exchange. The pure bioconjugate was injectedinto mice (n=10). Serum of mice that were immunized with O1-AcrA (top)or O1-EPA (bottom) three times (day 1, 21, 60) was pooled and analyzedby ELISA at day 70 for a sugar specific IgG response. The plates (Nunc,polysorb) were coated with LPS isolated from S. dysenteriae O1 andincubated with the serum and anti mouse Polyvalent-HRP. Mice thatreceived either conjugate developed an IgG response against thepolysaccharide, confirming the presence of T-cell epitopes on the twoprotein carriers.

Consequently, the bacterial bioconjugates of the present invention showin vivo immnogenicity. In an embodiment, bacterial bioconjugates arecapable of exhibiting: (1) a carbohydrate specific response; and (2) acarrier specific response or a similar response irrespective of thecarrier protein. Moreover, an IgG specific response shows T-celldependency of the immune response, such that memory of the response isexpected.

FIG. 12 reflects production of a Shigella O1 bioconjugate, e.g., O1-EPA,in a bioreactor. E. coli cells expressing EPA, PgIB and theO1-polysaccharide were grown in a bioreactor to OD₆₀₀=40 by two nutrientpulses. Expression of PgIB and EPA was induced and the cells were grownovernight by linear feed of nutrients. The growth curve is depicted inthe top panel. Whole cell extracts were separated by SDS-PAGE andexpression and glycosylation of EPA was analyzed by immunodetectionusing a polyclonal antierserum that was raised against EPA (bottom). Thecells efficiently glycosylate EPA at high cell density. The process isreproducible and leads to a total optical density (OD) of 90, which is a45-fold increase compared to the shake flask culture. Consequently,scale-up is possible using the fed-batch process.

FIG. 13 shows an example of purification of O1-EPA. More specifically,FIG. 13 shows the fractionation and chromatographic purification of S.dysenteriae O1 bioconjugate. E. coli cells expressing the O1-EPA weregrown in the bioreactor to high cell density (See FIG. 12). The cellswere pelleted by centrifugation and periplasmic proteins were extractedby osmotic shock. Periplasmic proteins were separated by anionicexchange (Source Q). Fractions enriched for O1-EPA were further purifiedby a second column (Fluoroapatite). The different fractions wereseparated by SDS-PAGE and the proteins were visualized by CoomassieBlue. Lane 1 shows whole cells extracts, lane 2 periplasmic proteinsafter osmotic shock, lane 3 periplasmic proteins loaded on anionicexchange, lane 4 and 5 eluates from anionic exchange and lane 6 O1-EPAeluate after the second purification column. This process allows thepurification of O1-EPA at large scale. In this embodiment, thepurification process is: (1) efficient (at >10 mg/L culture); (2)possible at large scale; and (3) compatible with Good ManufacturingPractices (GMP). Following such purification, the EPA-O1 yield forglycerol-LB fed-batch was up to 200 mg/L, which is substantially higherthan the yield for LB shake flask, which was 0.6 mg/L.

FIG. 14A shows a Western blot analysis of a series of specimens ofAcrA-Shigella O1 bioconjugates produced in an LB shake flask taken undervarious conditions, including pre-induction, 4 hours after induction,and 4 hours and 19 hours after induction under oxygen-limitedcircumstances. After extraction and purification, periplasmic proteinswere separated by SDS-PAGE. AcrA and AcrA-Shigella O1 bioconjugates weredetected using anti-AcrA antibody and chemiluminescent detection via asecondary antibody. Loaded samples were normalized to culture OD₆₀₀ attime of sampling.

In summary, in one aspect, the technology of the present invention hasbeen used to develop a vaccine against S. dysenteriae O1 infection. Forexample, the polysaccharide of S. dysenteriae O1 can be conjugated toEPA in E. coli. This is very beneficial since EPA previously has beensuccessfully used in clinical trials with different conjugate vaccines.In the instant invention, the S. dysenteriae O1 bioconjugate wasproduced in a bioreactor at 31 scale. The cells were grown to high ODand the bioconjugate was extracted by osmotic shock. The bioconjugateswere purified to 98% purity by anionic exchange and size exclusionchromatography. The bioconjugates were injected into different micestrains. After two as well as three injections, a sugar specific IgGresponse against the polysaccharide was detected using LPS from Shigelladysenteriae O1 for analysis (FIG. 11). As expected, IgM specificresponse was elicited when the LPS was injected. The bioconjugatesraised a specific IgG response against the polysaccharide isolated fromS. dysenteriae. IgG response against the corresponding sugar antigen,which was chemically coupled to a carrier protein, has been shown tocorrelate with protection in humans.

These results strongly suggest that our inventive E. coli strain issuitable for the potential production of an antigenic bacterial vaccine,such as an antigenic Shigella vaccine. In an embodiment, theEPA-Shigella bioconjugate was characterized intensively by differentmethods, like NMR, HPLC and MS. FIG. 15 shows the expansion of theanomeric region of 1H NMR spectrum of an example of a S. dysenteriaeSerotype O1 bioconjugate of the invention. The bioconjugate contains asugar/protein ratio of 0.15, with 13.2 repeating units of the antigenbeing linked to the protein, and 1-2 sites being glycosylated. Twoconsensus sequences for glycosylation were introduced into EPA and about20% of the protein is fully glycoslyated. The polysaccharide is linkedvia the reducing end to the protein carrier; therefore, the antigenepitopes of the polysaccharide are unmodified. In addition, the in vivoconjugation method attaches just the O-antigen repeating units to theprotein, but no monosaccharides of the lipid A core are attached.

Using this technology, bacterial bioconjugates can be produced that areimmunogenic. Genetic modifications can be made allowing in vivoconjugation of bacterial polysaccharides in desired proteins and atdesired positions. For example, in an embodiment and as discussed above,the antigenic polysaccharide of S. dysenteriae O1 can be expressed in E.coli and conjugated to two different protein carriers in vivo (i.e., EPAand AcrA). Both bioconjugates elicit a specific IgG response against thepolysaccharide in mice. As another example, Table 1 below depictsdifferent polysaccharide substrates for bacterial OSTs/OTases such asPgIB that can be used in the in vivo method of the present invention forconjugating a protein carrier with the polysaccharide.

TABLE 1 C. jejuni N-glycan

Shigella dysenteriae O1

Pseudomonas aeruginosa O11

E. coli O16

Table 2 below depicts yet additional different LPS polysaccharidesubstrates that could be utilized in the present invention with respectto various strains of Shigella and E. coli., as well as of Pseudomonasaeruginosa O11 and Francisella tularensis.

TABLE 2 Shigella dysenteriae O1

S. flexneri 2a

S. flexneri 3a

S. flexneri 3b

S. flexneri 6

S. sonnei

E. coli O4: K52 (ExPEC)

E. coli O4: K6 (ExPEC)

E. coli O6: K2 (ExPEC)

E. coli O6: K54 (ExPEC)

E. coli O22 (ExPEC)

E. coli O75 (ExPEC)

E. coli O83 (ExPEC)

E. coli O7

E. coli O9

E. coli O16

E. coli O121

E. coli 0157

Pseudomonas aeruginosa O11

Francisella tularensis

For example, in a further embodiment of the invention, bioconjugatevaccines against E. coli can also be developed. E. coli is a well-knownbacterial species. From a genetic and clinical perspective, E. colistrains of biological significance to humans can be broadly categorizedas commensal strains, intestinal pathogenic strains and extraintestinalpathogenic E. coli (ExPEC). ExPEC strains can be part of the normalintestinal flora and are isolated in 11% of healthy individuals. They donot cause gastroenteritis in humans but their main feature is theircapacity to colonize extraintestinal sites and to induce infections indiverse organs or anatomical sites. They are the main cause of urinarytract infections (UTI), are involved in septicemia, diverse abdominalinfections and meningitis. Bacteremia can arise with a risk of severesepsis. Severe sepsis due to ExPEC was associated with 41,000 estimateddeaths in 2001. ExPEC strains have been susceptible to antibiotics;however more and more antibiotic resistant strains have evolved, both inhospital and in the community. This antimicrobial resistance is makingthe management of ExPEC infections more difficult; therefore, newvaccines would be an alternative strategy to prevent these infections.

In animal models, passive or active immunization against capsule,O-specific antigen and different outer membrane proteins have affordedprotection against systemic infections and immunization with thesedifferent antigens are protective against urinary tract infections fromExPEC strains expressing these virulence factors. The serotypes O4, O6,O14, O22, O75 and O83 cause 75% of UTI. In one embodiment, the noveltechnology of the present invention can be used to develop a monovalentLPS bioconjugate including one antigen (e.g., serotype O6, one of themajor serotypes) and even a multivalent LPS bioconjugate including these6 antigens. For example, the gene cluster encoding for the enzymes thatsynthesize the O-antigen for ExPEC could be amplified and then expressedin the Shigella production strain.

The instant invention involves a highly efficient production processwith high potential yields that can be used for industrial scalepreparations in a cost-efficient process. This novel, cost efficientbioengineering approach to producing bioconjugate can be applied toother conjugates and for other applications. An additional feature ofthe invention involves a considerable simplification of the productionof bacterial vaccines with high reproducibility and a potentiallyreduced risk of lot failures.

Process for Manufacturing Conjugate Vaccine

It is now possible to engineer bacterial expression systems so thatspecific bioconjugates are produced that are biologically active. Forexample, the O-specific polysaccharide of S. dysenteriae has beenconjugated to different protein carriers and the resulting bioconjugatehas elicited a specific IgG response against the polysaccharide in mice.In an embodiment, the technology of the invention makes use of anoligosaccharyl transferase, for example, PgIB of Campylobacter jejuni tocouple bacterial polysaccharides (O antigens) in vivo to simultaneouslyexpress recombinant carrier proteins, yielding highly immunogenicbioconjugate vaccines.

A production process has been established that can be used on anindustrial scale. This opens up the possibility that a multitude ofvarious conjugate vaccines can be developed and manufactured usingsimple bacterial fermentation. The process has several advantagescompared to the in vitro conjugation method depicted in the top panel ofFIG. 6A. As it is a complete in vivo process, the cost and risk offailures are reduced significantly and the process is more reproducible.In addition, the consensus capture sequence allows the conjugation ofpolysaccharides to defined proteins at specific built-in sites, therebyfacilitating regulatory acceptance and quality control. Finally, thedevelopment of conjugate vaccines is much faster since the process issimplified and requires only biotechnology tools. In addition, the invivo conjugation process is suited for application where polysaccharidecompositions prevent chemical cross-linking.

In an embodiment, the instant invention relates to the scaled-upproduction of recombinant glycosylated proteins in bacteria and factorsdetermining glycosylation efficiency. For example, recombinantglycosylated proteins of the present invention can be made using theshakeflask process. Bioconjugates have previously been mainly producedin LB shake flask cultures. More preferably, in one aspect of theinvention, a first fed-batch process can be used for the production ofrecombinant glycosylated proteins in bacteria. In a preferredmanufacturing process, the aim is to achieve markedly increased finalbiomass concentrations while maintaining glycosylation efficiency andrecombinant protein yield per cell and while maintaining simplicity andreproducibility in the process.

In one embodiment, bacterial bioconjugates of the present invention canbe manufactured on a commercial scale by developing an optimizedmanufacturing method using typical E. coli production processes. First,one can use various types of feed strategies, such as batch, chemostatand fed-batch. Second, one can use a process that requires oxygen supplyand one that does not require an oxygen supply. Third, one can vary themanner in which the induction occurs in the system to allow for maximumyield of product.

It has found been that, in contrast to expression of the carrierprotein, the degree of N-linked glycosylation strongly reacts to changesin nutrient availability, type of carbon- and energy source, oxygensupply and time-point of induction. For example, in a fed-batch process,the addition of inducers to the batch and fed-batch cultures immediatelyleads to a 3-fold decrease in specific growth rate, indicating a highmetabolic burden and/or stress due to synthesis of the carrier proteinand membrane-bound oligosaccharyltransferase. Based on the inventors'finding of a recurring retardation of the appearance of glycosylatedcarrier protein compared to the non-glycosylated form after induction,it is concluded that glycosylation appears to be the rate-limiting stepin bioconjugate biosynthesis.

Based on these results, in an example of an embodiment of the invention,the following process design for cultivation has been developed:fed-batch cultivation mode for achieving high cell densities; extendedincubation after induction to facilitate maximal glycosylation; surplusnutrient supply (e.g., LB components yeast extract and tryptone) duringbiomass build-up until induction to provide a sufficient supply ofbuilding blocks for the production process; and glycerol as the maincarbon and energy source to prevent catabolite repression and acetateformation. This bioprocess allows a 50-fold increase in yield comparedto LB batch culture, paving the way towards a cost-effective productionof conjugate vaccines in recombinant Escherichia coli. In this example,one can have oxic conditions throughout the production process, forexample, achieved through oxygen-enriched aeration; however, low oxygencontent is also feasible. Example 9 sets forth this example of afed-batch process in greater detail. It should be recognized, however,that other processes may be used to produce the bacterial LPSbioconjugates of the present invention.

Consequently, in one embodiment of the invention, E. coli can be usedfor in vivo production of glycosylated proteins and is suitable forindustrial production of glycosylated proteins.

The following examples serve to illustrate further the present inventionand are not intended to limits its scope in any way.

EXAMPLES Example 1: Selection of AcrA as Model Protein for OptimizingN-Glycosylation

To optimize the acceptor protein requirements for N-glycosylationdetailed studies were performed on the C. jejuni glycoprotein AcrA(CjO367c). AcrA is a periplasmic lipoprotein of 350 amino acid residues.It has been shown that secretion to the periplasm but notlipid-anchoring is a prerequisite for glycosylation (Nita-Lazar et al.,2005, supra). The signal for export can either be the native AcrA signalsequence or the heterologous PeIB signal when expressed in E. coli. Ofthe five potential Winked glycosylation sequons (N117, N123, N147, N273,N274) the same two ones are used in C. jejuni and E. coli (N123 and N273(Nita-Lazar et al., 2005, supra)). AcrA was chosen as model because itis the only periplasmic N-glycoprotein of C. jejuni for which detailedstructural information is available. Recently, the crystal structure ofan AcrA homologue, the MexA protein from the Gram-negative bacterium P.aeruginosa, was published (Higgins et al., (2004). Structure of theperiplasmic component of a bacterial drug efflux pump. Proc. Natl. Acad.Sci. USA 7Of₁ 9994-9999). Both proteins are members of the so-calledperiplasmic efflux pump proteins (PEP,(Johnson, J. M. and Church, G. M.(1999). Alignment and structure prediction of divergent proteinfamilies: periplasmic and outer membrane proteins of bacterial effluxpumps. J. Mol. Biol. 287, 695-715)). The elongated molecule containsthree linearly arranged subdomains: an α-helical, anti-parallelcoiled-coil which is held together at the base by a lipoyl domain, whichis followed by a six-stranded β-barrel domain. The 23-28 residues at theN-terminus and 95-101 residues in the C-terminus are unstructured in thecrystals. MexA and AcrA protein sequences are 29.3% identical and 50%similar. Thus, the two proteins likely exhibit a similar overall fold.

Example 2: Elucidation of the Primary Peptide Sequence that TriggersGlycosylation

It is known that lipoyl domains similar to MexA of P. aeruginosa andaccordingly also in AcrA of C. jejuni form a compact protein that can beindividually expressed in E. coli (reviewed by Berg, A., and de Kok, A.(1997). 2-Oxo acid dehydrogenase multienzyme complexes. The central roleof the lipoyl domain. Biol. Chem. 378, 617-634). To check which acceptorpeptide sequence was required for N-glycosylation by the pgl machineryin E. coli the lipoyl domain of AcrA was taken. It was used as amolecular scaffold to transport peptides of different lengths to theperiplasm and present them to the pgl machinery in vivo.

Therefore, a plasmid coding for the lipoyl domain (Lip) was constructedand N-terminally fused to the signal sequence of OmpA (Choi, J. H., andLee, S. Y. (2004). Secretory and extracellular production of recombinantproteins using Escherichia coli. Appl Microbiol Biotechnol 64, 625-635)and C-terminally to a hexa histag. Cloning was performed to place thegene expression under the control of the arabinose promoter. For the Lipdomain borders amino acid positions were chosen that appeared at thesame positions as the domain borders of the Lipoyl domain part in MexA.To test different peptides for their ability to accept an N-glycanstretches of the sequence were inserted between the two hammerhead-likeparts of the Lip domain. The stretches consisted of sequences comprisingthe N-glycosylation site N123 of C. jejuni AcrA. The resulting openreading frames consisted of the sequences coding for the OmpA signalsequence, the N-terminal hammerhead-like part of AcrA (D60-D95, thenumbering of the amino acids refers to the mature AcrA polypeptidesequence numbering), the different stretches containing the native N123glycosylation site of AcrA (see below), the C-terminal hammerhead-likepart of AcrA-Lip (L167-D210) and the C-terminal his-tag.

Construction of the plasmids was achieved by standard molecular biologytechniques. Three stretches containing the native N123 glycosylationsite of AcrA of different lengths were inserted between the two halvesof Lip resulting in three different ORFs:

Construct A contains A118-S130 resulting in a protein sequence of:

(SEQ. ID NO. 8) MKKTAIAIAVALAGFATVAQADVIIKPQVSGVIVNKLFKAGDKVKKGQTLFIIEQDQASKDFNRSKALFSQLDHTEIKAPFDGTIGDALVNIGDYVSASTTELVRVTNLNPIYADGSHHHHHH.

Construct B contains F122-E138 resulting in a protein sequence of:

(SEQ. ID NO. 9) MKKTAIAIAVALAGFATVAQADVIIKPQVSGVIVNKLFKAGDKVKKGQTLFIIEQDQ FNRSKALFSQSAISQKELDHTEIKAPFDGTIGDALVNIGDYVSASTTELVRVTNLNPIYADGSHHHHHH.

Construct C contains D121-A127 resulting in a protein sequence of:

(SEQ. ID. NO. 10) MKKTAIAIAVALAGFATVAQADVIIKPQVSGVIVNKLFKAGDKVKKGQTLFIIEQDQDFNRSKALDHTEIKAPFDGTIGDALVNIGDYVSASTT ELVRVTNLNPIYADGSHHHHHH.

The underlined stretches of sequence indicate the OmpA signal peptide,singly underlined residues were introduced for cloning reasons or torender the protein resistant to degradation. Bold: glycosylation sitecorresponding to N123 of AcrA. Italics: hexa-histag. The correspondinggenes were expressed under the control of the arabinose promoter in thebackbone of the plasmid pEC415 (Schulz, H., Hennecke, H., andThony-Meyer, L. (1998). Prototype of a heme chaperone essential forcytochrome c maturation. Science 281, 1197-1200).

To check which of the three stretches triggered glycosylation of the Lipproteins protein expression experiments were performed. E. coli Top 10cells (Invitrogen, Carlsbad, Calif., USA) carrying pACYCpgl orpACYCpglmut (Wacker et al., 2002, supra) and a plasmid coding constructsA₁ B or C were grown in LB medium containing ampicillin andchloramphenicol up to an OD of 0.5 at 37° C. For induction 1/1000 volume20% arabinose (w/v) solution was added and the cells were grown foranother 2 hrs. The cells were then harvested by centrifugation andresuspended in 20 mM Tris/HCl, pH 8.5, 20% sucrose (w/v), 1 mM EDTA, 1mM PMSF, and 1 g/l (w/v) lysozyme and incubated at 4° C. for 1 hr.Periplasmic extracts were obtained after pelletting of the spheroblastsand diluted with 1/9 volume (v/v) of 10× buffer A (3 M NaCl, 0.5 MTris/HCl, pH 8.0 and 0.1 M imidazole) and MgSO₄ added to 2.5 mM.Ni-affinity purification was performed on 1 ml Ni-Sepharose columns fromAmersham Pharmacia Biotech (Uppsala, Sweden) in buffer A. Proteins wereeluted in buffer A containing 0.25 M imidazole.

FIG. 1 shows Coomassie brilliant blue stained SDS-PAGE gel of the peakelution fractions from the Ni-purified periplasmic extracts. Theexpression analysis showed that construct B produced a prominent singleprotein species (FIG. 1, lane 1). Constructs A and C both lead, inaddition to the prominent protein, to a second protein band with slowerelectrophoretic mobility (FIG. 1, lanes 2 and 3). That the heavierprotein species was indeed glycosylated was proven by MALDI-TOF/TOF (notshown). The only amino acid missing in construct B but present in A andC was D121, the aspartate residue 2 positions N-terminally to theglycosylated N123. This demonstrates that D121 plays an important rolefor glycosylation by the OTase. To verify that D121 is essential forglycosylation it was mutated to alanine in construct C. Expressionanalysis resulted in only one protein band (FIG. 1, lane 4), thusshowing that D121 is important for glycosylation. Furthermore, the factthat an artificial peptide display protein can be glycosylated showsthat a short peptide of the D/E-X-N-Y-S/T type contains all informationfor C. jejuni-borne N-glycosylation to occur.

Example 3: Verification of Example 2; AcrA-D121A is not Glycosylated atN123

To confirm the findings from the peptide display approach an aspartateto alanine mutation was inserted at position 121 (D121A, i.e. 2 residuesbefore the glycosylated N123) in the full length soluble version of theAcrA protein and it was tested whether the site N123 could still beglycosylated in E. coli. In order to test this AcrA-D121A was expressedand its glycosylation status was analyzed. For the analysis anengineered AcrA was used. It differed from the original C. jejuni genein that it contains the PeIB signal sequence (Choi and Lee, 2004, supra)for secretion into the periplasm and a C-terminal hexa histag forpurification. It has been shown that this AcrA variant gets secreted,signal peptide-cleaved and glycosylated as the lipid anchored, nativeprotein (Nita-Lazar et al., 2005, supra). The following is the aminoacid sequence of the soluble AcrA protein:

(SEQ. ID NO. 11) MKYLLPTAAAGLLLLAAQPAMAMHMSKEEAPKIQMPPQPVTTMSAKSEDLPLS/TYPAKLVSDYDVIIKPQVSGVIVNKLFKAGDKVKKGQTLFIIEQDKFKASVDSAYGQALMAKATFENASKDFNRSKALFSKSAISQKEYDSSLATFNNSKASLASARAQLANARIDLDHTEIKAPFDGTIGDALVNIGDYVSASTTELVRVTNLNPIYADFFISDTDKLNLVRNTQSGKWDLDSIHANLNLNGETVQGKLYFIDSVIDANSGTVKAKAVFDNNNSTLLPGAFATITSEGFIQKNGFKVPQIGVKQDQNDVYVLLVKNGKVEKSSVHISYQNNEYAIIDKGLQNGDKIILDNFKKIQVGSEVKEIGAQLEHHHHHH

The underlined residues are the PelB signal peptide, italics thehexa-histag, and bold the two natural glycosylation sites at N123 andN273. A plasmid containing the ORF for the above protein in the pEC415plasmid (Schulz et al., 1998) was constructed to produce pAcrAper.

The assay to test the glycosylation status of AcrA and mutants thereof(see below) was as follows: expression of AcrA was induced with 0.02%arabinose in exponentially growing E. coli CLM24 (Feldman et al., 2005,supra) cells containing the plasmid-borne pgl operon in its active orinactive form (pACYCpg/or pACYCpg/mut, see (Wacker et al., 2002, supra))and a plasmid coding for AcrA (pAcrAper). After four hours of induction,periplasmic extracts were prepared as described above and analyzed bySDS-PAGE, electrotransfer and immunodetection with either anti-AcrAantiserum or R12 antiserum. The latter is specific for C. jejuniN-glycan containing proteins (Wacker et al., 2002, supra).

The first two lanes of FIG. 2A show AcrA in the absence and presence ofa functional pgl operon. Only one band appears in the absence but threein the presence of the functional pgl operon (FIG. 2A, top panel). Thesecorrespond to unglycosylated AcrA (lane 1) and un-, monO- anddiglycosylated AcrA (lane 2). That the two heavier proteins in lane 2were glycosylated was confirmed by the R12 western blot (lane 2, bottompanel). When the mutant AcrA-N273Q was expressed the same way, only themonoglycosylated AcrA was detected in presence of the functionalglycosylation pgl operon (lane 3). Unglycosylated AcrA was detected inabsence of the functional pgl locus (lane 4). Analysis of the mutantAcrA-D121A produced only two bands, one of them glycosylated (lane 5) asobserved with AcrA-N273Q in lane 3. This means that D121 is essentialfor efficient glycosylation at position 123-125.

Example 4: Introducing Artificial Glycosylation Sites into AcrA

To test if the introduction of an aspartate residue could generate aglycosylation site, AcrA mutants were generated in which the residue inthe −2 position of the not used glycosylation sites in positions N117and N147 of soluble AcrA were exchanged for aspartate (F115D, T145D). Itwas then tested whether the modified glycosylation sites could beglycosylated by the same assay as described in example 2. Both mutationswere individually inserted either into the wildtype sequence of thesoluble version of AcrA or in the double mutant in which both usedglycosylation sites were deleted (N123Q and N273Q). Periplasms extractsof cultures induced for 4 hrs were prepared, separated by SDS page andanalyzed by Western blotting (FIG. 2B). As controls the samples ofwildtype glycosylated and non glycosylated AcrA were run on the same gel(lanes 1 and 2). The T145D mutation affected the −2 position of thenatively not used glycosylation sequon N147-S149. Upon expression ofAcrA-T145D Western blotting with anti AcrA antiserum resulted in fourbands, the highest of them with slower electrophoretic mobility than thedoubly glycosylated protein in lane 2 (lane 3 in FIG. 2B). The R12 blotconfirmed that the fourth band was a triply glycosylated AcrA. Despitethe low intensity towards anti AcrA the heaviest band gave the strongestsignal with the glycosylation specific R12 antiserum. When the samemutant AcrA-T145D was expressed in the absence of the nativeN-glycosylation sequence (AcrA-N123Q-N273Q-T145D), only monoglycosylatedAcrA was detected in the presence of a functional pgl operon (FIG. 2B,lane 4), that was missing in absence of a functional pgl operon (lane5). This demonstrates that the heavier band in lane 4 was glycosylated.Hence, by simply introducing the T145D mutation an optimizedglycosylation site was generated (DFNNS).

To further confirm that it is possible to introduce a glycosylation siteby inserting an aspartate residue in the −2 position, the natively notused sites N117-S119 and N274-T276 were changed to optimizeN-glycosylation. For this purpose further mutants were generated (FIG.2C). Expression of AcrA-F115D-T145D in the above described systemresulted in five protein species detected with the anti AcrA antiserum(lane 2). This is indicative for four glycosylates taking place on thesame AcrA molecule. When the detection was performed with the C. jejuniN-glycan-specific R12 antiserum, a ladder of five bands was detected.The lowest faint band is unglycosylated AcrA because it is also presentin the absence of glycosylation (lane 1), the highest results in astrong signal probably due to the five antigenic determinants in afourfold glycosylated AcrA. Thus, the two introduced sites (at NI 17 andN147) and the two natively used sites (N123 and N273) are used andglycosylated by the pgl machinery. Expression of AcrA-N123Q-N273Q-N272Dwith and without the pgl operon demonstrated that a third artificiallyintroduced glycosylation site, N274 (DNNST), was also recognized by thepgl operon (FIG. 2C, lanes 3 and 4).

The above experiments confirm the finding that the bacterialN-glycosylation site recognized by the OTase of C. jejuni consistspartly of the same consensus as the eukaryotic one (N-X-S/T, with X≠P)but, in addition, an aspartate in the −2 position is required forincreasing efficiency. Furthermore, they demonstrate that it is possibleto glycosylate a protein at a desired site by recombinantly introducingsuch an optimized consensus sequence.

Example 5: Verification of Position −1 in the Optimized N-GlycosylationSequence

A further experiment was performed to test whether the −1 position inthe bacterial glycosylation site exhibits the same restrictions as the+1 position in eukaryotes (Imperiali, B., and Shannon, K. L. (1991).Differences between Asn-Xaa-Thr-containing peptides: a comparison ofsolution conformation and substrate behaviour witholigosaccharyl-transferase. Biochemistry 30, 4374-4380; Rudd, P. M., andDwek, R. A. (1997). Glycosylation: heterogeneity and the 3D structure ofproteins. Crit. Rev. Biochem. Mol. Biol. 32, 1-100). A proline residueat +1 is thought to restrict the peptide in such a way thatglycosylation is inhibited. To test if a similar effect could also beobserved in the −1 position a proline residue was introduced at thatposition of the first natively used site in a point mutant that had thesecond native site knocked out (AcrA-N273Q-F122P). The controlexpression of AcrA-N273Q showed a monoglycosylated protein in thepresence of a functional pgl operon (FIG. 2D, lane 1 and 2). However,AcrA-N273Q-F122P was not glycosylated (FIG. 2D, lanes 3 and 4). Thisindicates that proline inhibited bacterial N-glycosylation when itconstitutes the residue between the asparagine and the negativelycharged residue of the −2 position.

Sequence alignments of all the sites known to be glycosylated by the C.jejuni pgl machinery indicate that they all comprise a D or E in the −2position (Nita-Lazar et al., 2005, supra; Wacker et al., 2002, supra;Young et al., (2002). Structure of the N-linked glycan present onmultiple glycoproteins in the Gram-negative bacterium, Campylobacterjejuni. J. Biol. Chem. 277, 42530-42539). Thus, it was established thatthe glycosylation consensus sequence for bacteria can be optimized by anegatively charged amino acid in the −2 position, resulting inD/E-X-N-Z-S/T, wherein X & Z≠P.

Example 6: N-Glycosylation of a Non-C. jejuni Protein

To demonstrate that the primary sequence requirement (optimizedconsensus sequence) is sufficient for N-glycosylation in bacteria, itwas tested whether a non-C. jejuni protein could be glycosylated byapplying the above strategy. Cholera toxin B subunit (CtxB) was employedas a glycosylation target. The corresponding gene was amplified fromVibrio cholerae in such a way that it contained the coding sequence ofthe OmpA signal sequence on the N-terminus and a hexahistag at theC-terminus, just the same as constructs A through C in example 1. Theresulting DNA was cloned to replace construct A in the plasmids employedin example 1. A point mutation of W88 to D or a D insertion after W88generated an optimized glycosylation site (DNNKT). The wildtype and W88DCtxB proteins containing the signal sequence and his-tag were expressedin E. coli Top 10 and other cell types in the presence and absence ofthe functional pgl locus from C. jejuni. When periplasmic extracts fromTop 10 cells were analyzed by SDS-PAGE, electrotransfer and consecutiveimmunoblotting with a CtxB antiserum, only CtxB W88D produced a higherand thus glycosylated band in the pgl locus background (FIG. 2E, comparelanes 3 and 4). A consensus sequence (DSNIT) was also inserted byreplacing G54 or Q56 of CtxB (the latter is denoted CtxB-Q56/DSNIT),i.e. in one of the loops that was reported to contribute to theganglioside GM 1 binding activity of CtxB. Lanes 5 and 6 of FIG. 2Edemonstrate that the engineered protein (exemplified by the constructwhich contains the peptide sequence DSNIT instead of Q56 expressed inTop10 cells) produced a lower mobility and thus glycosylated band inglycosylation competent but not glycosylation-deficient cells whenanalyzed in the same way as described above. It was also demonstratedthat a CtxB containing two manipulations, i.e. the insertion of D afterW88 as well as DSNIT replacing Q56, was double-glycosylated in SCM7cells (Alaimo et al., EMBO Journal 25: 967-976 (2006)) (panel E, lanes 7and 8). The double-glycosylated protein CtxB shown in lane 7 was Ni²⁺affinity-purified and analyzed by ESI-MS/MS after in-gel trypsinizationaccording to standard protocols. The expected glycopeptides weredetected confirming that bacterial N-glycosylation can also be directedto a non-C. jejuni protein by mutating or inserting the optimizedconsensus sequence according to the invention for bacterialN-glycosylation (not shown). Examples of other suitable exemplary E.coli strains for practicing the present invention are W3110, CLM24, BL21(Stratagene, La Jolla, Calif., USA), SCM6 and SCM7.

The amino acid sequence of the CtxB protein used here is indicated below(recombinant OmpA signal sequence underlined, hexa-histag italics, W88bold):

(SEQ. ID NO. 12) MKKTAIAIAVALAGFATVAQATPQNITDLCAEYHNTQIHTLNDKIFSYTESLAGKREMAIITFKNGATFQVEVPGSQHIDSQKKAIERMKDTLRIAYLTEAKVEKLCVWNNKTPHAIAAISMANGSHHHHHH

Example 7: Introduction of Artificial N-Glycosylation Sites into the C.jejuni Outer Membrane Protein, OmpH1

A potential application of the N-glycosylation in bacteria is thedisplay of the glycan on the surface of a bacterial host cell in orderto link the phenO- to the genotype and thereby select for specificgenetic mutations. To demonstrate that N-glycans can be presented onouter membrane proteins the OmpH1 protein was engineered in a way thatit contained multiple optimized consensus sites according to theinvention. The sites were engineered into loop regions of the protein asdeduced from the known crystal structure (Muller, A., Thomas, G. H.,Horler, R., Brannigan, J. A., Blagova, E., Levdikov, V. M., Fogg, M. J.,Wilson, K. S., and Wilkinson, A. J. 2005. An ATP-binding cassette-typecysteine transporter in Campylobacter jejuni inferred from the structureof an extracytoplasmic solute receptor protein. Mol. Microbiol. 57:143-155). Previous experiments showed that the best glycosylationsequons were generated by the mutations V83T, K59N-G601-N61T,R190N-G191I-D192T and H263D-F264S-G265N-D2661-D267T. For surface displayit was desired to evaluate different combinations of those introducedsites in order to establish the most N-glycan-specific sample. Thecombinations were generated in a wild type OmpH1 encoding plasmidconstruct and tested in a similar manner as described for AcrA. FIG. 3shows the analysis of various OmpH1 variants harboring multipleglycosylation sequons in addition to the existing wild type sequon.OmpH1 variants were generated with three (lane 3, 4, 5 and 7) and fourglycosylation sequons (lane 6). A wild type OmpH1 with only oneglycosylation sequon and a mutant lacking the critical asparagine forglycosylation were also included in the experiment. All variants testedhere did not only demonstrate a high level of glycosylation efficiencybut also that every glycosylation sequon was utilized. The results wereconfirmed with Campylobacter N-glycan specific immuneserum (FIG. 3 lowerpanel).

The following is the amino acid sequence of the OmpH1 protein ofCampylobacter jejuni (strain 81-176) with attached myc tag in italics:

(SEQ. ID NO. 13) MKKILLSVLTTFVAVVLAACGGNSDSKTLNSLDKIKQNGWRIGVFGDKPPFGYVDEKGNNQGYDIALAKRIAKELFGDENKVQFVLVEAANRVEFLKSNKVDIILANFTQTPERAEQVDFCLPYMKVALGVAVPKDSNITSVEDLKDKTLLLNKGTTADAYFTQDYPNIKTLKYDQNTETFAALMDKRGDALSHDNTLLFAWVKDHPDFKMGIKELGNKDVIAPAVKKGDKELKEFIDNLIIKLGQEQFFHKAYDETLKAHFGDDVKADDWIEGGKILEQKL/SEEDL

The native glycosylation site in the protein is bold, the signalsequence underlined.

Example 8: Surface Display of N-Glycans from C. jejuni on OmpH1 on theOuter Membrane of E. coli Cells

In order to answer the question whether multiple glycosylated OmpH1variants can be displayed on the surface of bacterial cells,immunofluorescence was performed on bacterial CLM24 or SCM6 (which isSCM7 ΔwaaL) cells expressing various OmpH1 variants. A wild type OmpH1and a mutant lacking the critical asparagine for glycosylation wereincluded in the experiment. In addition, a C20S mutant was constructedin order to retain the protein in the periplasm, thus serving as acontrol in the experiment. Immunostaining was carried out on the cellstreated with paraformaldehyde. Paraformaldehyde fixes cells withoutdestroying the cell structure or compartimentalization. The c-Myc- andN-glycan-specific immuneserum in combination with correspondingsecondary antibodies conjugated to FITC and Cy3 were used to detect theprotein (red fluorescence) and N-glycan (green) on the bacterial cellsurface, respectively. Additionally, 4,6-diaminO-2-phenylindole (DAPI,blue) was employed to stain for bacterial DNA to unambiguouslydifferentiate between bacterial cells and cellular debris. When thecells expressing wild type OmpH1 were stained, immunofluorescencespecific to the protein as well as the N-glycan was detected (FIG. 4 A).When a mutant lacking the critical asparagine N139S was stained withboth anti-Myc- and N-glycan-specific immuneserum only the protein butnot glycan specific signals were obtained (panel 4 B) indicatingspecificity of the N-glycan-specific immune serum. When the protein wasretained within the periplasm as in the C20S mutant, no proteinspecific, red immunofluorescence was detected indicating that theantibodies were unable to diffuse within the cell and were competentenough to detect any surface phenomenon (panel 4 C). Next, cellsexpressing multiple OmpH1 variants different in glycosylation werestained: OmpH1^(KGN→NIT.,HFGDD→DSNIT) (panel 4 D),OmpH1^(RGD→NIT),^(HFGDD→DSNIT) (panel 4 E), OmpH1^(KGN→NIT,RGD→NIT)(panel 4 F), OmpH1^(V83T,KGN→NIT) (panel 4 G) andOmpH1^(KGN→NIT),^(RGD→NIT HFGDD→DSNIT) (panel 4 H). All the OmpH1variants were double-stained indicating the presence of glycosylatedprotein on the bacterial surface. FIG. 4 is represented in grayscale,the first column is a merge picture of the other pictures of the samerow.

FIG. 4 shows fluorescence microscopy of cells expressing various OmpH1variants. Cultures of E. coli strains CLM24 or SCM6 containing theexpression plasmid for the wild type OmpH1 and its variants wereequalized to OD₆₀₀ of 0.25/ml. Cells were washed two times withphosphate-buffered saline (PBS), pH 7.4 and 100 μl cell suspensions wasdropped onto gelatinized glass slides and incubated at room temperature(RT) for 30 min inside a humidified chamber. All subsequent steps in thewhole-cell immunofluorescence labeling were done at room temperatureinside a humidified chamber. The unbound cells were removed and rest wasfixed with 4% paraformaldehyde containing PBS for 30 min at RT.Importantly, paraformaldehyde is considered not to permeabilize cellsbut keeping the compartimentalization by membranes intact. Fixed cellswere washed two times with PBS and resuspended blocking buffercontaining 5% BSA in PBS. After blocking, the cells were incubated withanti-myc monoclonal mouse IgG (1:50, Calbiochem) and/or anti-glycanantiserum (1:4000) for 1 h in 100 μl of PBS containing 5% BSA. The cellswere washed three times with 100 μl of PBS for 5 min each and incubatedwith secondary anti-rabbit antibody conjugated to FITC (1:250, JacksonImmunoresearch Laboratories) and/or anti-mouse antibody conjugated toCy3 (1:250, Jackson Immunoresearch Laboratories) for 1 h in 100 μl ofPBS containing 5% BSA. If required, 4,6-diaminO-2-phenylindole (DAPI)(Sigma) (0.5 μg/ml) was added at the time of secondary antibodyincubation to stain for bacterial DNA. The secondary antibody was rinsedfrom the cells PBS₁ and coverslips were mounted on slides by usingvectashield (Vector Laboratories) mounting medium and sealed with nailpolish. Fluorescence microscopy was performed by the using an Axioplan2microscope (Carl Zeiss). Images were combined by using Adobe Photoshop,version CS2. SCM6 cells expressing OmpH1 (panel A), OmpH1^(N139S) (panelB), OmpH1^(C20S) (panel C), OmpH1^(KGN→NIT, HFGDD→DSNIT) (panel D),OmpH1^(RGD→NIT,HFGDD→DSNIT) (panel E), OmpH1^(KGN→NIT RGD→NIT) (panelF), OmpH1^(V83T,KGN→NIT) (panel G), andOmpH1^(KGN→NIT,RGD→NIT,HFGDD→DSNIT) (panel H). The first column is amerge of the pictures in columns 2, 3, and 4 represented in greytones onblack background. Column 2: blue fluorescence in greytones from DAPIstain, column 3: green fluorescence from glycan specific fluorescence,column 4: red fluorescence from anti-myc staining.

Example 9: An Example of a Production Process for Shigella O1 LPSBioconjugate

This is an example of a production process; however, differentconditions also lead to similar product formation.

A. Production Process

E. coli strain W3110ΔwaaL containing three plasmids expressing PglB, EPAand the enzymes for the biosynthesis of the Shigella O1 polysaccharidewas used for the production of the LPS bioconjugate. A single colony wasinoculated in 50 ml LB medium and grown at 37° C. O/N. The culture wasused to inoculate a 11 culture in a 21 bioreactor. The bioreactor wasstirred with 500 rpm, pH was kept at 7.0 by autO-controlled addition ofeither 2 M KOH or 20% H₃PO₄ and the cultivation temperature was set at37° C. The level of dissolved oxygen (pO2) was kept between 0 and 10%oxygen. The cells were grown in a semi defined glycerol mediumcontaining Kanamycin to an OD₆₀₀=15. The medium contained the followingingredients: 330 mM Glycerol, 10 g Yeast extract, 20 g Tryptone, 34 mMK₂HPO₄, 22 mM KH₂PO₄, 38 mM (NH₄)₂SO₄, 2 mM MgSO₄.7H₂O and 5 mM Citricacid. After an initial batch phase around 5 h, a first nutrient pulsewas added to sustain fast biomass build-up (glycerol, tryptone and yeastextract). After an additional 1.5 h the culture reached an OD₆₀₀=30. Atthis timepoint a second nutrient pulse of glycerol and tryptone wasadded together with the required inducers 1% L-arabinose and 1 mM IPTG.In order to keep induction at maximum levels and supply further aminoacids for recombinant protein synthesis, a linear nutrient/inducer feed(28.8 ml/h) was started with the addition this pulse. The feed wassustained until the end of the process. The bioreactor culture washarvested after a total of 24 h cultivation, when it should have reachedan OD600 of ±80.

The production process was analyzed by Western blot as describedpreviously (Wacker, M., et at, N-linked glycosylation in Campylobacterjejuni and its functional transfer into E. coli. Science, 2002.298(5599): p. 1790-3.). After being blotted on nitrocellulose membrane,the sample was immunostained with the specific anti-EPA (Wacker, M., etal., N-linked glycosylation in Campylobacter jejuni and its functionaltransfer into E. coli. Science, 2002. 298(5599): p. 1790-3.).Anti-rabbit IgG-HRP (Biorad) was used as secondary antibody. Detectionwas carried out with ECL™ Western Blotting Detection Reagents (AmershamBiosciences, Little Chalfont Buchinghamshire).

FIG. 16A shows proteins extracted of the Shigella O1 LPS Bioconjugate(i.e., EPA-O1) from a fed-batch process that were normalized to biomassconcentration (0.1 OD_(600 nm) of cells/lane). The proteins wereseparated by SDS-PAGE transferred to Nitrocellulose membrane andvisualized by rabbit anti EPA antibody. The induction time for PglB andEPA expression was 1 h and O/N.

B. Periplasmic Protein Extraction

The cells were harvested by centrifugation for 20 min at 10,000 g andresuspended in 1 volume 0.9% NaCl. The cells were pelleted bycentrifugation during 25-30 min at 7,000 g. The cells were resuspendedin Suspension Buffer (25% Sucrose, 100 mM EDTA

200 mM Tris HCl pH 8.5, 250 OD/ml) and the suspension was incubatedunder stirring at 4-8° C. during 30 min. The suspension was centrifugedat 4-8° C. during 30 min at 7,000-10,000 g. The supernatant wasdiscarded, the cells were resuspended in the same volume ice cold 20 mMTris HCl pH 8.5 and incubated under stirring at 4-8° C. during 30 min.The spheroblasts were centrifuged at 4-8° C. during 25-30 min at 10,000g, the supernatant was collected and passed through a 0.2μ membrane.

As shown in FIG. 16B, the periplasmic extract was loaded on a 7.5%SDS-PAGE, and stained with Coomasie to identify EPA and EPA-O1. EPA is athick band that runs above the 70 kDa marker. O1-EPA (i.e., EPA-O1) runsas a leader between 100 and 170 kDa.

C. Bioconjugate Purification

The supernatant containing periplasmic proteins obtained from 100,000 ODof cells was loaded on a Source Q anionic exchange column (XK 26/40≈180ml bed material) equilibrated with buffer A (20 mM Tris HCl pH 8.0).After washing with 5 column volumes (CV) buffer A, the proteins wereeluted with a linear gradient of 15CV to 50% buffer B (20 mM Tris HCl+1MNaCl pH 8.0) and then 2CV to 100% buffer B. Protein were analyzed bySDS-PAGE and stained by Coomassie. Fractions containing O1-EPA werepooled. Normally the bioconjugate eluted at conductivity between 6-17mS. The sample was concentrated 10 times and the buffer was exchanged to20 mM Tris HCl pH 8.0.

As shown in FIG. 17A, protein fractions from 1. Source Q were analyzedby SDS-PAGE and stained by Coomassie. Fractions C1 to G9 contained O1bioconjugate and were pooled.

The O1-Bioconjugate was loaded a second time on a Source Q column (XK16/20≈28 ml bed material) that has been equilibrated with buffer A: 20mM Tris HCl pH 8.0. The identical gradient that was used above was usedto elute the bioconjugate. Protein were analyzed by SDS-PAGE and stainedby Coomassie. Fractions containing O1-EPA were pooled. Normally thebioconjugate eluted at conductivity between 6-17 mS. The sample wasconcentrated 10 times and the buffer was exchanged to 20 mM Tris HCl pH8.0.

As shown in FIG. 17B, protein fractions from 2. Source Q column wereanalyzed on SDS-PAGE and stained by Coomassie. Fractions A11 to B3containing O1 bioconjugate were pooled.

The O1-Biconjugate was loaded on Superdex 200 (Hi Load 26/60, prepgrade) that was equilibrated with 20 mM Tris HCl pH 8.0.

As shown in FIG. 18A, protein fractions from Superdex 200 column wereanalyzed by SDS-PAGE and stained by Coomassie stained. Fractions F1 toF11 were pooled.

As shown in FIG. 18B, Shigella bioconjugate from different purificationsteps were analyzed by SDS-PAGE and stained by Coomassie. O1-EPA waspurified to more than 98% purity using the process, showing that O1-EPAbioconjugate can be successfully produced using this technology.

Example 10: Engineering of Exotoxin a of Pseudomonas aeruginosa forGlycosylation with Antigenic Carbohydrates

Exotoxin A of Pseudomonas aeruginosa (EPA) is a 67 kDa extracellularysecreted protein encoding mature 613 amino acids in its mature form andcontaining four disulfide bridges (C11-C15, C197-C214, C265-C287,C372-C379). To enable its glycosylation in E. coli, the protein mustlocate to the periplasmic space for glycosylation to occur. Therefore, athe signal peptide of the protein DsbA from E. coli was geneticallyfused to the N-terminus of the mature EPA sequence. A plasmid derivedfrom pEC415 [Schulz, H., Hennecke, H., and Thony-Meyer, L., Prototype ofa heme chaperone essential for cytochrome c maturation, Science, 281,1197-1200, 1998] containing the DsbA signal peptide code followed by aRNase sequence was digested (NdeI to EcoRI) to keep the DsbA signal andremove the RNase insert. EPA was amplified using PCR (forward oligo was5′-AAGCTAGCGCCGCCGAGGAAGCCTTCGACC (SEQ. ID NO. 14) and reverse oligo was5′-AAGAATTCTCAGTGGTGGTGGTGGTGGTGCTTCAGGTCCTCGCGCGGCGG (SEQ. ID NO. 15))and digested NheI/EcoRI and ligated to replace the RNase sequenceremoved previously. The resulting construct (pGVXN69) encoded a proteinproduct with an DsbA signal peptide, the mature EPA sequence and ahexa-histag. Detoxification was achieved by mutating/deleting thecatalytically essential residues L552VΔE553 according to [Lukac, M.,Pier, G. B., and Collier, R. J., Toxoid of Pseudomonas aeruginosaexotoxin A generated by deletion of an active-site residue, InfectImmun, 56, 3095-3098, 1988] and [Ho, M. M., et al., Preclinicallaboratory evaluation of a bivalent Staphylococcus aureussaccharide-exotoxin A protein conjugate vaccine, Hum Vaccin, 2, 89-98,2006] using quick change mutagenesis (Stratagene) and phosphorylatedoligonucleotides 5′-GAAGGCGGGCGCGTGACCATTCTCGGC (SEQ. ID NO. 16) and5′-GCCGAGAATGGTCACGCGCCCGCCTTC (SEQ. ID NO. 17) resulting in constructpGVXN70.

It is known that insertion of a pentapeptide sequence of the typeD/E-Z-N-X-S/T into a suitable position results in glycosylation. Toglycosylate EPA in E. coli cells, two different glycosylation sites wereinserted into the previously described constructs according to thefollowing description.

To insert a site at position 375, two steps were performed. First, quickchange mutagenesis using oligos 5′-CCTGACCTGCCCCGGGGAATGCGCGG (SEQ. IDNO. 18) and 5′-CCGCGCATTCCCCGGGGCAGGTCAGG (SEQ. ID NO. 19) with pGVXN70as a template resulted in a construct containing a single SmaI site atamino acid position 375 of EPA protein sequence by deleting threeresidues but otherwise keeping the starting protein sequence intact. Ina second step, an insert composed of two complementary, phosphorylatedoligonucleotides coding for (i) the previously deleted residues (wheninserting the SmaI site), (ii) the pentapeptide glycosylation sequon and(iii) additional lysine residues flanking the consensus for optimizationof glycosylation efficiency (as was found by further experiments) wasligated into this SmaI site (5′-GTCGCCAAAGATCAAAATAGAACTAAA (SEQ. ID NO.20) and 5′-TTTAGTTCTATTTTGATCTTTGGCGAC (SEQ. ID NO. 21). The resultingconstruct was pGVXN137.

To insert an additional glycosylation site in the construct at aminoacid 240, a one step procedure using quick change mutagenesis witholigonucleotides5′-CATGACCTGGACATCAAGGATAATAATAATTCTACTCCCACGGTCATCAGTCATC (SEQ. ID NO.22) and 5′-GATGACTGATGACCGTGGGAGTAGAATTATTATTATCCTTGATGTCCAGGTCATG (SEQ.ID NO. 23) was applied on construct pGVXN137. The resulting constructthus contained various changes compared to the wild type EPA protein:two glycosylation sites, a DsbA signal peptide, detoxification mutation.

While this invention has been particularly shown and described withreferences to embodiments thereof, it will be understood by thoseskilled in the art that various changes in form and detail may be madetherein without departing from the scope of the invention encompassed bythe claims. Moreover, in instances in the specification where specificnucleotide or amino acid sequences are noted, it will be understood thatthe present invention encompasses homologous sequences that still embodythe same functionality as the noted sequences. Preferably, suchsequences are at least 85% homologous. More preferably, such sequencesare at least 90% homologous. Most preferably, such sequences are atleast 95% homologous.

The invention claimed is:
 1. A method of producing a compositioncomprising a bioconjugate separated from periplasmic proteins, whereinsaid bioconjugate comprises a carrier protein linked to an O antigen,said method comprising: (i) culturing a prokaryotic host cell thatcomprises (A) a nucleic acid encoding a carrier protein comprising theamino acid sequence D/E-X-N-Z-S/T, wherein X and Z may be independentlyany natural amino acid except proline; (B) a nucleic acid encoding anoligosaccharyl transferase; and (C) one or more nucleic acids encodingone or more glycosyltransferases capable of assembling the O antigen ona lipid carrier, wherein said culturing is done for a period of timesufficient for the host cell to produce the bioconjugate; (ii)extracting periplasmic proteins from the prokaryotic host cell; and(iii) separating the bioconjugate from the extracted periplasmicproteins and unconjugated carrier protein by anionic exchange, whereinsaid O antigen is from extraintestinal pathogenic Escherichia coli(ExPEC), Shigella flexneri 2a, S. flexneri 3a, S. flexneri 3b, S.flexneri 6 or S. sonnei.
 2. The method of claim 1, wherein said0-antigen is heterologous to said host cell.
 3. The method of claim 1,wherein said carrier protein is heterologous to said host cell.
 4. Themethod of claim 1, wherein said carrier protein is Pseudomonasaeruginosa exoprotein (EPA); AcrA (CjO367c), HisJ (CjO734c), or OmpH1(CjO982c) from Campylobacter jejuni; Diphteria toxin (CRM197); orCholera toxin.
 5. The method of claim 4, wherein said carrier protein isPseudomonas aeruginosa exoprotein (EPA).
 6. The method of claim 1,wherein said oligosaccharyl transferase is heterologous to said hostcell.
 7. The method of claim 1, wherein said oligosaccharyl transferaseis from Campylobacter.
 8. The method of claim 7, wherein saidoligosaccharyl transferase is from Campylobacter jejuni.
 9. The methodof claim 1, wherein said composition is at least 98% pure with respectto the bioconjugate.